Dna-cell conjugates

ABSTRACT

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/263,129, filed Feb. 24, 2012, which is the U.S. National Stage ofInternational Application No. PCT/US2010/030397, filed Apr. 8, 2010,which claims priority to U.S. Application No. 61/167,748 filed Apr. 8,2009, and 61/243,123 filed Sep. 16, 2009, all of which are incorporatedherein in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with Government support under Contract No.DE-AC02-05CH11231 awarded by the U.S. Department of Energy and supportedby the Nanoscale Science, Engineering, and Technology (NSET) Program ofthe Department of Energy, and the Office of Science, Office of BasicEnergy Sciences, under Grant Nos. HG003329 and R01 GM072700 awarded bythe National Institutes of Health, and under a National Institutes ofHealth Molecular Biophysics Training Grant No. T32GM08295. TheGovernment has certain rights in this invention

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

This application includes a Sequence Listing as a text file namedSEQTXT_077429-008111US-1003079 modified Feb. 26, 2016 and containing6,732 bytes. The material contained in this text file is incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Typically, peptides have been used in cell-based arrays to capture cellsfor study. Surfaces are printed with “RGD” peptides designed to bindintegrins on a cell surface, but this system does not work with cellsthat don't have integrins such as non-adherent cells (e.g. leukocytes,lymphocytes) and does not allow controlled defined patterning withdifferent cell types together in the same platform. The RGD system hasthe large disadvantage of initiating cell differentiation, thus changingthe cell before it is analyzed, because integrins are the receptors thatare involved in controlling differentiation and also activities thatfollow after integrin binding that relate to differentiation. (See Du,X. P. et al., Cell 1991, 65, 409-416; Xiong, J. P. et al., Science 2002,296, 151-155.)

Indirect noncovalent attachment is demonstrated with a protein-proteinattachment system in which DNA is indirectly attached to cells by anoncovalent linkage through an antibody-ligand interaction. (Bailey R Cet al. J Am Chem Soc. 2007 February 21; 129(7):1959-67) An antibodyspecific for a target ligand on a cell is conjugated to DNA. Thenoncovalent linkage between the antibody and the ligand is based onhydrogen bonding, typical of protein-protein interactions.Single-stranded DNA (ssDNA) oligomers on antibodies specific forcell-surface ligands are attached to cells having those ligands, and thecells are in turn anchored to surfaces having ssDNA complementaryoligomers that bind the partner strand on the cell to capture the cell.

Indirect covalent attachment of synthetic single-stranded DNA (ssDNA)strands to the surfaces of living cells was first shown using metabolicoligosaccharide engineering by Chandra, R. A. et al. Angew. Chem.-Int.Edit. 2006, 45, 896-901. The indirect covalency was through specificchemical handles (azides) that were introduced in cell surface sialicacids obtained after treating the cells with peracetylatedN-azidoacetylmannosamine (Ac4ManNAz) (taking 3 days) prior to theintroduction of the DNA. Phosphine-ssDNA conjugates were then covalentlyattached to the azide handle to form an amide bond (a Staudingerligation reaction, E. Saxon, et al. Science 2000, 287) between theazido-sugar and the DNA. Azides installed within cell surfaceglycoconjugates by metabolism of a synthetic azidosugar can be reactedwith a biotinylated triarylphosphine to produce many stable cell-surfaceadducts. However, the covalent metabolic approach takes multiple days toprepare the cells for DNA attachment, and altering the cell surfacesugars has metabolic effects on the cell that changes it before one getsa chance to analyze it. It is further limited to certain mammalian cellsthat possess sialic acid on their surface, and thus cannot be used forbacteria, plant cells, fungi, or many other animal cells.

Noncovalent attachment via antibodies and ligands at the cell surfaceswill also activate the cells and thus perturb the cell before analysiscan start, also tending to be weaker and “reversible” compared with acovalent attachment. In addition the antibody mechanism requiresprevalence of ligand on the cell, and engineering an antibody specificfor the ligand to affix sufficient DNA on the cell surface.

Noncovalent attachments via ligand interactions with an antibody at thecell-surface have been made where the antibody carries a strand ofprotein binding DNA. (Bailey R C et al. J Am Chem Soc. 2007 February 21;129(7):1959-67). Both methods have the immediate disadvantage ofactivating the cell they seek to capture for study, thus transformingthe thing of interest into something different before it can beanalyzed. Overcoming the drawbacks in these early systems of DNAattachment on cell surfaces could transform this just described nacentfield and offer valuable tools and manipulations previously notpossible.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a composition having acell, wherein the cell has a surface including a native functionalgroup, and wherein the cell has no cell wall. The composition alsoincludes a nucleic acid moiety, wherein the nucleic acid moiety iscovalently linked to the native functional group.

In another embodiment, the present invention provides a method ofpreparing a conjugate of a cell and a nucleic acid moiety by contactingthe cell with an activated nucleic acid moiety, wherein the cell has asurface including a native functional group, and wherein the cell has nocell wall, such that the nucleic acid moiety is covalently linked to thenative functional group.

In another embodiment, the present invention provides a compositionhaving a cell, wherein the cell has a cell wall, and a nucleic acidmoiety, wherein the nucleic acid moiety is covalently linked to thecell.

In another embodiments, the present invention provides a method ofpreparing a conjugate of a cell and a nucleic acid moiety, by contactingthe cell with an activated nucleic acid moiety, wherein the cellincludes a cell wall, such that the nucleic acid moiety is linked to thecell.

In another embodiment, the present invention provides a device includinga cell having a cell surface of a native functional group covalentlylinked to a first nucleic acid moiety, and a substrate surface having asecond nucleic acid moiety complementary to the first nucleic acidmoiety, such that the cell is bound to the substrate surface viaformation of a nucleic acid duplex of the first and second nucleic acidmoieties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Covalent attachment of ssDNA to cell surfaces. (a) Thiolatedsingle-stranded DNA was first reacted with NHS-PEG-Maleimide in PBS atroom temperature to form the NHS-DNA conjugate. This solution was thenincubated with suspensions of live cells in PBS at room temperature for30 mins. After attachment of the DNA strands, the cells were returned toculture media. (b,c) Jurkat cells were exposed to NHS-DNA solutions ofvarying concentrations as described in (a). The fluorescent strandcomplement was then added, and the level of cell modification wasquantified using flow cytometry. Up to 120,000 DNA strands could beinstalled on each cell. (d) A schematic of a method for patterning cellsonto a surface.

FIG. 2. Immobilization of cells in a DNA sequence-specific manner. (a)Cells bearing DNA sequence C2 bound to complementary (sequence M2) spotson a DNA microarray (spot size=60 μm). Neighboring spots withnon-complementary sequence M1 remained unoccupied. (b) The samemicroarray substrate was exposed to a mixed suspension of Jurkat and MDAcells bearing sequences C2 and C1, respectively. Jurkat cells werestained with Cell-Tracker Green, and MDA cells were stained withCell-tracker Blue. (c) MCF-7 and (d) MDA cells also bound to DNA coatedsurfaces in a rapid, stable, and sequence-specific manner. Cleardelineation between the DNA-coated and uncoated regions was observed.Phase contrast images are shown after 2 h of incubation. (e) MCF-7 and(f) MDA cells had spread and proliferated after 36 h, but were stillconfined to the DNA-printed area.

FIG. 3. Direct DNA modification and capture of primary cells. (a) Humanred blood cells were bound in the same manner as Jurkat cells on a DNAspot, and appeared to be morphologically identical immediately afterbinding. Trypan blue staining indicated that the membranes remainedintact. (b) DNA-coated mouse CD4+ helper T cells were bound by spotscoated with complementary DNA. After 3 minutes of exposure, a clearboundary can be seen between the printed and unprinted regions of theslide. (c) Microscale DNA patterns made by photolithography andmicrofabrication. Fluoresein-conjugated ssDNA strands were patterned onthe substrate to allow visualization. (d) Mouse primary T cells werecaptured on the same DNA patterns. (e) IL-2 production ofDNA-immobilized T cells and free T cells, as determined by ELISA.ConA=concanavalin A. PMA=phorbol meristyl acetate. CSA=cyclosporin A.

FIG. 4. Capture and differentiation of primary myoblast cells. (a,b) DNApatterns on glass slides dictate the areas in which cells are bound. (c)Myoblast cells show no signs of differentiation immediately aftercapture (shown), or after 1 day when kept in growth media. (d) Myotubesform upon addition of differentiation media. The photo was taken fivedays after the switch was made. (e) After 6 days of incubation indifferentiation media, circularly patterned myoblasts form arcedmyotubes that are aligned with the edge. (f) After 6 days, myocytes inrectangular arrangements form myotubes that are aligned with the longaxis of the patterns. For the linear patterns, the majority of themyocytes (g) align to within 20° of the pattern boundary angle and (h)are found half way between the edges.

FIG. 5. Matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF) mass spectrometry of DNA modification reactions. A modelamine compound was found to react with the NHS ester, verifying theformation of amides on the cell surfaces.

FIG. 6. Viability of Jurkat cells modified with NHS-DNA. A) A solutionof DNA-coated cells (20 bases each) was combined with the complementarystrands. At various time points the total number of cells was countedvisually (Blue curve). The control sample (orange curve) consisted ofunmodified Jurkat cells grown in the absence of added DNA. B) Toevaluate viability after attachment, DNA-modified cells were immobilizedon glass slides bearing the strand complement. After immobilization for24 h and 48 h, the cells was incubated with a solution of annexin V-FITC(green bars) and PI (red bars). The cells were evaluated within 1 husing fluorescence microscopy. Free cells were control samples thatlacked surface DNA and were not bound to the slides.

FIG. 7. Immobilized MCF-7 and MDA cells were imaged after 2, 12, 24, 36hours.

FIG. 8. Myoblasts were incubated in growth media for 3 days. A)Myoblasts were seeded on collagen coated dishes. B) Myoblasts werereacted with NHS-DNA and bound to the surface in a sequence-specificmanner.

FIG. 9. Myoblasts incubated in fusion media after 2, 4, 6, 8, 10, 12, 14days and either seeded on collagen coated dishes or reacted with NHS-DNAand bound to the surface in a sequence-specific manner.

FIG. 10. Analysis of myotube alignment on a defined pattern. Thedistance (red line) and angles were measured between the myotubes andthe nearest edge. All distance measurements were made from the midpointof the tubes. The full distance between the pattern edges is indicatedby the green line.

FIG. 11. Fabrication of the bifunctional microelectrode array for singlecell monitoring. (A) Gold electrodes are patterned on a glass waferusing photolithography and liftoff. A 7 μm insulating layer ofParylene-c is then deposited onto the electrodes, and covered with a 100nm layer of evaporated aluminum. (B) Photoresist is patterned on thealuminum layer, which is then etched and used as an etch mask for theParylene insulation. (C) The sensor layer of iridium oxide is depositedon the electrode surface and then treated with an aldehyde silane foramine-modified capture DNA attachment. (D) Finally, the aluminum layeris dissolved in strong base, leaving only the capture DNA on the sensorsurface. Cells bearing the surface-bound complementary strand areintroduced and captured directly and specifically on the sensor. (E)Cells are treated with single stranded DNA (5′-CCCTAGAGTGAGTCGTATGA-3′;SEQ ID NO:4) bearing a terminal N-hydroxysuccinimidyl (NHS) esterfunctional group, which binds to primary amines on the cell surface.This DNA barcode labeling functionalizes the cell for DNA-directedcapture in the device. (F) Schematic of the microfluidic device. Theelectrodes are enclosed by a PDMS channel, forming the microfluidicdevice.

FIG. 12. Calibration data for the bifunctional microelectrode array. (A)Typical calibration recording for one DNA-modified iridium oxide sensorusing standard pH 4, 5 and 7 buffers. Voltage is measured relative to anAg/AgCl reference electrode. (B) Plot of the voltage vs. pH standardmeasurement with a slope of −68.5 mV/pH unit and R²=0.99995.

FIG. 13. Cell capture on the bifunctional microelectrode array.Fluorescent micrograph of individual non-adherent Jurkat cells with asurface-bound DNA barcode bound to the complementary strand on thesensor electrode. Electrode areas are outlined in white. Bar=40 μm.Inset: Magnified view of a single Jurkat cell on an electrode, withadditional oblique illumination to reveal the electrode area.

FIG. 14. Single cell acidification measured with the bifunctionalmicroelectrode array. (A) Representative composite data of single Jurkatand primary T cell acidification measured in known homogenous samples.(B) Single Jurkat and primary T cells captured from a mixture andmonitored simultaneously over a 10 min span on the array. (C) Histogramof individual cell acidification in known-type samples over 10 min.Jurkat cells are seen to have a significantly higher (P<0.0002) rate ofacidification than primary T cells in low-buffered media.

FIG. 15. Single cell stimulation measured by the bifunctionalmicroelectrode array. Jurkat cells exhibit normal baseline acidificationduring the first 13 minutes, then 125 μL of 10 μM rotenone inlow-buffered media is added to the channel outlet reservoir where itdiffuses into the channel within seconds. Rotenone inhibits themitochondrial electron transport chain, causing an increased rate oflactic acid excretion, and therefore a higher rate of acidification.

FIG. 16. Covalent attachment of biomolecules to cantilevers and cellsurfaces. a) After surface oxidation using an oxygen plasma, aldehydefunctional groups were introduced onto silicon nitride cantilevers usingchemical vapor deposition (CVD). b) Solutions of anti-CD3 IgG or ConAcontaining sodium borohydride were introduced onto aldehyde-coatedcantilever surfaces in a humid chamber (IgG=immunoglobulin G). DNAmodification was achieved by immersing cantilevers in anamine-functionalized ssDNA solution at 100° C. for 30 min and subsequentexposure to a sodium borohydride solution. c) Metabolic engineering wasused to introduce azide groups onto cell surfaces by treatment withperacetylated N-azidoacetylmannosamine (Ac₄ManNAz).Phosphine-functionalized ssDNAs were synthesized and covalently attachedto the exterior of cells by Staudinger ligation.

FIG. 17. Comparison of biomolecule-based adhesion methods. a) Bulk cellgrowth rates were first determined in the presence of the adhesionmolecules. A suspension of Jurkat cells was combined with ConA oranti-CD3 IgG, and a solution of DNA-coated cells was combined with thecomplementary DNA strands. At various time points the total number ofcells was counted. The control sample was grown in the absence of anyadhesion molecules. b) To evaluate cell capture efficiency, solutions of20 μM FITC-labeled ssDNA, 20 μM FITC-labeled ConA, and 6 μM FITC-labeledanti-CD3 IgG were applied to aldehyde-coated glass slides, and thebiomolecules were attached by reductive amination. Solutions containing1×10⁷ Jurkat cell mL⁻¹ were then introduced onto the resulting slides.The samples were incubated for 10 min at room temperature and thenwashed with two portions of phosphate-buffered saline (PBS) beforeevaluation. c) To evaluate cell viability, cells were immobilized onDNA, ConA, and anti-CD3 IgG coated aldehyde slides. After immobilizationfor 24 and 48 h, the cells were incubated with a solution of annexinV-FITC (black bars) and PI (gray bars). The cells were evaluated within1 h by fluorescence microscopy. * ConA and antibody immobilized cellsthat were partially stained by annexin were counted as cells undergoingapoptosis. NB represents control samples that were not bound to thesurfaces. Error bars represent one standard deviation.

FIG. 18. AFM measurement of de-adhesion force. A) Six sample traces fora single cell are shown in shades of gray, with the average trace shownin black. At zero distance, the cell is in full contact with thecantilever, which is applying a positive force. As distance increases,the cantilever is pulled away from the glass slide surface, causgin thecell-cantilever linkage to rupture and result in the zero-force,no-contact region. The force of d-adhesion was calculated as thedifference between the curve minimum and the horizontal no=contactregion. B) Adhesion forces were measured under different retractionrates (15.7 and 8.2 μm/s) and contact forces (400 and 200 pN) for theDNA, ConA, and antibody systems. Data were obtained by measuring sizede-adhesion events on more than four different cells. Error bardsrepresent one standard deviation.

FIG. 19. Dip-pen patterning of live cells. a) By attaching a shorter DNAstrand (13 bases) to a cantilever and a longer strand (20 bases) to theglass slide, a single living cell can be transported by the AFMinstrument and directly printed at a desired location on the glassslide. b-d) This process shown stepwise for the formation of a singlepattern of cells.

FIG. 20. Overview of single-cell gene silencing assay. Jurkat cells arecultured and surface-labeled, a single cell is captured on a target padvia DNA duplex formation, and an RT-PCR expression profile is generated.(A) Cells under normal growth conditions exhibit homogenous highexpression of GAPDH (green cells) compared with a control 18S rRNA. (B)Cells treated with siRNA directed at GAPDH exhibit varying levels ofmRNA knockdown.

FIG. 21. Microfluidic device layout. Schematic showing half of thedevice (2 of the 4 complete systems) for single-cell gene expressionprofiling. The 4-layer glass-PDMS-glass-glass microdevice contains 4distinct regions. The first region at the top is a 3-valve pump. Thereactor region consists of a photolithographically defined goldcell-capture pad in the center of a 200-nL reaction chamber along withRTDs and a microfabricated heater for thermal cycling. The affinitycapture region comprises a hold chamber and an affinity capture chamber(yellow). Finally, the thermally released amplicons are analyzed on theCE separation channel (red). Each device contains 4 independentlyaddressable systems enabling the analysis of 4 single cells in parallel.All channels are etched to a depth of 20 μm

FIG. 22. Schematic of the biochemical steps performed in the integratedgene expression microdevice. (Upper) The analysis is complete in <75min. (Lower) (A) Depiction of the operation of the single-cell geneexpression microsystem. (B) First, cells functionalized with a 20-baseoligonucleotide on their cell membrane are flowed into the reactor. (C)A single cell is captured on a size-limiting 25-×25-μm² gold pad whenthe ssDNA on its exterior binds to the complementary capture strandimmobilized on the gold pad. (D) The immobilized cell is freeze-thawlysed, and mRNA is reverse-transcribed into a stable cDNA strand (15min). PCR amplification (30 cycles) is completed in 25 min. (F)Amplified fragments and unreacted RT-PCR mixture are pumped from thereactor into the hold chamber and electrophoretically driven from thewaste (W) to the cathode (C) reservoirs. (F) Fragments of interest withcomplementarity to the affinity capture probe are concentrated andimmobilized at the entrance of the capture chamber creating a purifiedcapture plug. (G) Finally, the products are thermally released at 80° C.from the affinity capture gel and electrophoretically separated as theymigrate toward the anode (A). Fluorescently labeled amplicons aredetected by confocal fluorescence to determine their amount and identity

FIG. 23. Gene expression and silencing at the single-cell level. (A)Representative gene expression electropherograms from individual Jurkatcells. A single wild-type cell with primers targeting GAPDH (200 bp) and18S rRNA (247 bp) generates 2 strong peaks migrating at 160 s and 185 s,respectively. A single cell electroporated with siRNA directed at GAPDHmRNA shows only a single peak for 18S rRNA. (B) Gene expression of GAPDHfor Jurkat cells treated with GAPDH siRNA relative to normal untreatedcells. GAPDH expression has been normalized to a control 18S rRNA forcomparison. Experiments from 8 individual cells show GAPDH mRNA levelsat 0, 5, 50, 1, 48, 0, 5, and 0% of normally untreated Jurkat cells.However, a representative bulk measurement from 50 cells shows GAPDHexpression at 21±4%. When no cell is captured on the pad there is noamplification. Similarly, a PCR control with no reverse transcriptaseshows no amplification. (C) Histogram of the number of events for siRNAtreated cells shows that there are 2 distinct populations of cells whoseexpression levels are very distinct from the population average

FIG. 24. Dual-surface modification of capsides for targeted delivery isshown. For interior surface modification, an N87C mutation of the MS2coat protein allows for site specific alkylation. Up to 180 cargomolecules can be installed in these locations. For exterior surfacemodification, the aptamer (SEQ ID NO:10) is first modified with aphenylene diamine group. A T19paF mutation on the capsid allows for theattachment of the modified DNA to the exterior surface of MS2 by NaIO₄mediated oxidative coupling reaction.

FIG. 25. Analysis of DNA attachment to the exterior surface of MS2. (a)MS2-DNA conjugates were analyzed by SDS-PAGE followed by Coomassiestaining. Lanes 1-7 were reacted with strand A. Lane 8 shows thereaction with strand B. (b) a gel-shift assay confirmed DNA competencyfor base-pairing after conjugation to MS2. Lane 10 includes thecomplementary sequence to strand A with an additional 20 adenine basesfor increased electrophoretic shift, while lane 9 has not additional DNAadded. Transmission electron micrograph images (c) and dynamic lightscattering analysis (d) showed intact capsides after DNA conjugation. Inaddition, DLS showed a significant increase in diameter upon conjugationof strand A, as well as an additional increase in diameter upon additionof the 20-base complementary sequence to strand A (without theadditional 20-base overhang). The scale in the TEM image represents 100nm.

FIG. 26. Cellular targeting and uptake with aptamer-labeled capsids (a)cell targeting was confirmed using flow cytometry. Only MS2 capsidsmodified with strand B bound to Jurkat cells (blue). Capsids that wereunmodified on the exterior and capsids modified with strand C (green andyellow respectively) show a colocalization of B-labeled capsides withLDL-labeled endosomes (b), but not with transferrin-labelled endosomes(c). Scale bars represent 3 μm.

FIG. 27 shows installation of interior cargo attachment ofcell-targeting aptamers to viral exterior. The schematic depicts thebasic process involving the virus: the virus is coated with anoligonucleotide aptamer at a carbohydrate, the virus contains cargo (upto 180 molecules); by virtue of the aptamer specificity for a cellsurface protein on the surface of a live target cell, the aptamer bindsthe receptor protein and internalizes or otherwise delivers its contentsto that cell.

FIG. 28 shows images of cells which have been patterned using thepresent methods and a short explanation of the organism's role in asynergistic solar powered H2 fuel cell (on a chip). FIG. 28A showsSynechocystis PCC6803 (cyanobacteria), C. reinhardtii (algae), R.rubrum, and FIG. 28B shows A. vinelandii. The cells are patterned onlarge spots of ssDNA, which is why only a partial curvature to thepattern is shown in the images.

FIG. 29 shows images of E. coli patterns (A) shows patterned E. coli 20×labeled with FITC DIC overlay; (B) and (C) show images of patterned E.coli (not fluorescently labeled) and Synechocystis (labeled with FITC))microarray pattern 20× (top) and 10× (bottom). There is backgroundfluorescence but the cells are denotable by their texture.

FIG. 30 shows a schematic of Synechocystis PCC6803 (cyanobacteria), A.vinelandii, and R. rubrum patterned layers on a glass substrate for usein a fuel cell device.

FIGS. 31A and 31B show the DNA-cell conjugates of mammalian cellsprepared by the present invention by reaction with a lysine nativefunctional group on the cell surface, and immobilized on a glassmicroscope slide modified with the complementary DNA strand,demonstrating formation of the DNA cell conjugate.

FIGS. 32A and 32B show the DNA-cell conjugates of non-mammalian cellsprepared by the present invention immobilized on a glass microscopeslide modified with the complementary DNA strand, demonstratingformation of the DNA-cell conjugate.

FIG. 33 shows the DNA-cell conjugates of mammalian cells prepared by thepresent invention by reaction with an aspartic acid or glutamic acidnative functional group on the cell surface, and immobilized on a glassmicroscope slide modified with the complementary DNA strand,demonstrating formation of the DNA-cell conjugate.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides the first example of a cell-DNA conjugateprepared by covalently linking the DNA to the cell via a nativefunctional group on the cell surface, without the need for metabolicengineering. For mammalian cells and other cells without cell walls, theDNA is covalently linked directly to the amino acids on the surface ofthe cell, such as the lysine amines. For plant cells and other cellswith cell walls, the sugars on the surface of the cell are firstmodified, such as by oxidation to form an aldehyde or ketone, and thenmodified with the DNA.

Previous methods of forming DNA-cell conjugates required the use ofmetabolic engineering, i.e., feeding a cell with an appropriatelymodified sugar, such as an azide functionalized sugar, that wasmetabolised by the cell and expressed on the cell surface. The DNA wasthen conjugated to the azide modified sugar. This method has severaldrawbacks limiting both the utility and scope of application. Forexample, the metabolic engineering requires several days to afford acell with a sufficient amount of azide functionalized sugar on the cellsurface. The type of cells that can be modified is also limited,excluding primary cells which have important diagnostic utility.Finally, the metabolic engineering necessarily modifies the structureand properties of the cell.

The present invention overcomes the difficulties inherent to the azidemetabolic engineering process by using the native functional groupsalready present on the cell surface. Using an activated DNA sequence,such as DNA modified with an NHS-ester, the DNA can be covalently linkedto the amine of a lysine group on the cell surface of a cell without acell wall. This process takes several hours at most. The simplicity ofthe process allows modification of primary cells, as well as stem cells.For cells with cell walls, the native sugars on the cell surface arefirst modified, such as by oxidation to form aldehydes and ketones thatare then reacted with an activated DNA sequence, such as with anaminooxy group. The DNA-cell conjugates and methods of the presentinvention represent a substantial leap forward in cell detection andassay methods.

The ability to pattern cells on surfaces provides a new platform forvarious studies and applications, e.g., the study of cell biology, thecontrol of stem cell differentiation, and the engineering of newtissues. Typically, cell-based arrays are formed by printing surfaces ofinterest with “RGD” peptides that are designed to bind to integrins onthe cell surface. While this approach has been widely adopted for theimmobilization of many cell types, it cannot be used to capturenon-adherent cells (such as leukocytes) or to bind multiple cell typesto unique array features. It also can cause undesired changes in celldifferentiation or behavior because it engages the very surfacereceptors that are involved in controlling these processes.

To circumvent these limitations, the capture of live cells through thehybridization of synthetic DNA strands covalently linked to their plasmamembranes to surfaces printed with complementary sequences has beenpreviously reported by some of the inventors in Chandra, R. A.; Douglas,E. S.; Mathies, R. A.; Bertozzi, C. R.; Francis, M. B. Angew. Chem.-Int.Edit. 2006, 45, 896-901, hereby incorporated by reference. In additionto allowing multiple cell types to be patterned on a single substrate,the method offered the important advantages of substrate reuse andtunability. Most importantly, this approach has been used to capturenon-adherent cells in addition to adherent ones, and it has been shownthat the cells experience minimal changes in behavior as a result ofimmobilization through this receptor-independent process. In previousreports, the utility of this method has also been shown for theformation of complex cell patterns.

The DNA strands used in previous studies were installed into cellsurface glycans through a two-step process. First, the cells were fedwith an azide-containing mannose derivative for 1-3 days. This sugar wassubsequently metabolized and incorporated into sialic acid-containingcell surface glycans. The DNA was then targeted to the azidefunctionality using a Staudinger ligation. While effective, thisprotocol is most appropriate for cultured mammalian cell lines, as itrequires multiple days of exposure to install a sufficient number ofazide groups.

To expand the generality of the DNA-based adhesion method, the presentinvention provides an improved method for the direct installation ofnucleic acids onto virtually any cell surface. Referring now to FIG. 1,in one embodiment, an activated or functionalized single-strandednucleic acid is first reacted with chemical linker in a buffer solutionto form a nucleic-acid-linker conjugate. The cell or cell surface isexposed to the buffer solution for a specified period of time to allowthe reaction to proceed to attach the nucleic acid via the chemicallinker to a cell surface. After attachment of the nucleic acid to thecells, the cells are returned to culture media. Varying concentrationsof the nucleic acid can be used as described and quantified in FIG. 1a .In one example, an oligonucleotide is reacted with a chemical linker ina neutral buffer solution to conjugate the oligonucleotide to thechemical linker, then the cells are incubated with the buffer solutioncontaining the oligonucleotide-linker conjugate for 30 minutes to allowmodification and attachment of the oligonucleotide-linker conjugate tothe cell's surface.

The cell modification typically proceeds through the formation of acovalent bond between the linker and an amino acid on the cell surface.In some embodiments, the cell surface may be the cell membrane of livecells from any origin, including animal, plant, algae, or bacterialcells. In some embodiments, the covalent bond is an amide bond or anester bond. In some embodiments, the oligonucleotide is single-stranded.In other embodiments, the amino acid is selected from lysine, cysteine,tyrosine, serine, aspartate, glutamate and tryptophan.

In cells such as plant cell with cell walls, the linkage can be ahydrazone, an oxime, or an amine, among others, wherein said attachmentoccurs through periodate oxidation followed by hydrazone, oxime or amineformation. In one embodiment, a carbohydrate on the cell undergoesoxidation to generate an aldehyde function group, then the aldehyde isreacted with the oligonucleotide-linker conjugate for direct cellmodification of the plant cell surface.

This procedure can be carried out in some embodiments in less than 1hour, and leads to equivalent levels of cell surface functionalizationwith any oligonucleotide sequence of interest. The present method can beapplied to various embodiments including, the capture of single cellsfor RT-PCR analysis, the attachment of living cells to a solid substratefor force measurement or cell patterning techniques. In the examples, wedemonstrate the use of this new labeling method for the capture of redblood cells, primary T-cells, and myoblasts, which are all types ofcells that are difficult to pattern using other methods. This newtechnique greatly expands the scope of the DNA-based adhesion strategyand is sufficiently straightforward to be used in labs that do notspecialize in organic synthesis.

Thus, in one embodiment, the invention is a composition including a cellmembrane with an oligonucleotide directly covalently attached. The cellmembrane can be a whole intact cell, whereby the composition comprisinga whole cell with an oligonucleotide directly covalently attached.Multiple oligonucleotides can be attached to a single cell. The cell canbe a live cell. The cell can be any cell, such as a eukaryotic cell,including an animal cell or a non-animal cell. That the cell or cellmembrane is modified “directly” means that the cell membrane (cellsurface, outside of the cell) is not modified or changed before theattachment of the oligonucleotide. Specifically, because the attachmentis to a constituent on the cell surface, directly means that theconstituent to which the oligonucleotide attaches is not modified beforethe covalent attachment with the oligonucleotide. Previous methods allmodify a moiety on the cell surface first, before attaching theoligonucleotide.

By covalent attachment it is meant that a new covalent bond is formedbetween the two molecules that connect to each other. The bonds aretypically an amide or an ester bond, but can be any bond that serves thepurpose of attachment between the oligonucleotide and a moiety on thecell surface (such as a protein, amino acid, or carbohydrate, or othercell surface entity).

One embodiment facilitates the direct modification of cell surfaces withNHS-DNA conjugates in a rapid and efficient process, allowing virtuallyany mammalian cell to be patterned on surfaces bearing complementary DNAin under 1 hour. The specific technique described here demonstrates anability to use several types of cells that are generally incompatiblewith previous integrin-targeting techniques, including red blood cells,primary T-cells, and myoblasts. The immobilization procedure did notactivate primary T-cells, in contrast to previously reported antibody-and lectin-based methods. In these studies, myoblast cells werepatterned with high efficiency and remained undifferentiated aftersurface attachment. Upon changing to differentiation media, myotubesformed in the center of the patterned areas with an excellent degree ofedge alignment. The availability of this new protocol greatly expandsthe applicability of the DNA-based attachment strategy for thegeneration of artificial tissues and the incorporation of living cellsinto device settings.

II. Definitions

“Cell” refers to the basic functional unit of life, and includes bothprokaryotic and eukaryotic cells. Cells are characterized by an interiorhaving the nucleus or nucleoid, and a cell membrane (cell surface).Cells can also have a cell wall. Cells without a cell wall includeeukaryotic cells, mammalian cells, and stem cells. Cells with a cellwall include prokaryotic cells and plant cells. Other cells are usefulin the present invention.

“Native functional group” refers to the functional groups that arenative to the surface of a cell, such as amino acids and sugars, andthat react with the nucleic acid moiety to form the conjugates of thepresent invention. Exemplary amino acids include lysine, cysteine,tyrosine, threonine, serine, aspartic acid, glutamic acid andtryptophan. Other amino acids are useful, such as those described below.Sugars are also native to cell surfaces, and include mannose, galactoseand sialic acid, as well as those described below. The native functionalgroups can react with the nucleic acid moieties in an unmodified form,or can be modified to make them more reactive.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

“Amino acid analogs” refers to compounds that have the same basicchemical structure as a naturally occurring amino acid, i.e., an acarbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, e.g., homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid.

“Unnatural amino acids” are not encoded by the genetic code and can, butdo not necessarily have the same basic structure as a naturallyoccurring amino acid. Unnatural amino acids include, but are not limitedto azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethyl asparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,ornithine, pentylglycine, pipecolic acid, thioproline,aminophenylalanine, hydroxytyrosine, and aminotyrosine.

“Amino acid mimetics” refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid(i.e., hydrophobic, hydrophilic, positively charged, neutral, negativelycharged). Exemplified hydrophobic amino acids include valine, leucine,isoleucine, methionine, phenylalanine, and tryptophan. Exemplifiedaromatic amino acids include phenylalanine, tyrosine and tryptophan.Exemplified aliphatic amino acids include serine and threonine.Exemplified basic aminoacids include lysine, arginine and histidine.Exemplified amino acids with carboxylate side-chains include aspartateand glutamate. Exemplified amino acids with carboxamide side chainsinclude asparagines and glutamine. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Sugar” refers to a saccharide, such as a monosaccharide, adisaccharide, an oligosaccharide or a polysaccharide. Monosaccharidesinclude, but are not limited to, glucose, ribose, fructose, sialic acid,mannose and galactose. Disaccharides include, but are not limited to,sucrose and lactose. Polysaccharides include, but are not limited to,cellulose, hemicellulose and lignocellulose or starch. Other saccharidesare useful in the present invention.

As used herein, the term “contacting” refers to the process of bringinginto contact at least two distinct species such that they can react. Itshould be appreciated, however, the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

“Nucleic acid moiety” refers to a group containing a plurality ofnucleotides or nucleic acids. Exemplary nucleic acid moieties include,but are not limited to, an oligonucleotide, deoxy-ribonucleic acid(DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), Morpholinoand locked nucleic acid (LNA), glycol nucleic acid (GNA), threosenucleic acid (TNA), single-stranded DNA (ssDNA), 2′-fluorodeoxyribonucleic acid, aptamer, and others.

“Activated nucleic acid moiety” refers to a nucleic acid moiety have agroup having increased reactivity with one or more native functionalgroups. For example, when the native functional group is an amine onlysine, the activated group can be an activated ester such as anN-hydroxysuccinimide ester (NHS-ester). The activated ester can belinked directly to the nucleic acid portion of the activated nucleicacid moiety, or linked via a linker. When the native functional group iscysteine, the activated group can be a maleimide. Other activated estersand activated groups are useful for the activated nucleic acid moiety.Tyrosines can be modified using diazonium salts, imines, andallyl-palladium species. Tryptophans can be modified usingmetallocarbenoids and imines. N-terminal amino acids can be modifiedthrough transamination, and N-terminal serines and threonines can beoxidized to yield aldehydes using sodium periodate.

“Modified native functional group” refers to a native functional groupmodified to bind the nucleic acid moiety to the cell surface. The nativefunctional group can be modified in a variety of methods, except bymetabolic engineering. For example, when the native functional is asugar, the sugar can be oxidized using a suitable modifying agent oroxidizing agent such as sodium periodate. When the native functionalgroup is a sugar, and the modifying agent is an oxidizing agent such assodium periodate, the modified native functional group can be analdehyde or ketone.

“Substrate surface” refers any material which can be derivatized toinclude a nucleic acid moiety. Examples of materials for the substratesurface include, but are not limited to, glass (includingcontrolled-pore glass), polymers (e.g., polystyrene, polyurethane,polystyrene-divinylbenzene copolymer), silicone rubber, quartz, latex, aderivatizable transition metal, magnetic materials, silicon dioxide,silicon nitride, gallium arsenide, and derivatives thereof. Except forthe reactive sites on the surface, the materials are generally resistantto the variety of chemical reaction conditions to which they may besubjected.

III. Cell-DNA Conjugates

The present invention provides cell-DNA conjugates and methods of makingthe conjugates.

A. Conjugates of Cells without Cell Walls

The present invention provides conjugates of nucleic acid moieties andcells without cell walls. The conjugates are formed by covalentlylinking the nucleic acid moiety to the cell surface via a nativefunctional group on the cell surface. The cells used in the conjugatesof the present invention can be any cell, and do not require metabolicengineering to introduce the functional group for conjugating thenucleic acid moiety.

In some embodiments, the present invention provides a conjugate of acell and a nucleic acid moiety, wherein the cell has a surface includinga native functional group, the cell has no cell wall, and the nucleicacid moiety is covalently linked to the native functional group.

Cells useful in the present invention include any type of cell. In someembodiments, the cells are cells without a cell wall. Cells (and cellmembranes for the same use) that can be used for nucleic acid moietyattachment can be any eukaryotic cell which includes all animal cells,plants cells, algae cells, bacterial cells and fungal cells. Thefollowing is a non-exhaustive list of cells that may be used in thecompositions, devices and methods of this invention. The list isintended to include many cells and cell types, but not intended to belimiting of the cells that can be used in the invention. Rather thesecells and cell types are exemplary, and illustrative. Both live cells,killed cells, and cell lines may be used in the invention. Live cellsare likely to provide the most analytical and useful information, andbecause live cells can be used in the invention without activatingthrough cell surface modification, they can be studied for their nativeor near native properties in artificial systems and devices using thetools provided by and inherent to the invention.

The following is a nonlimiting list of cells. Human cell types fromblood and immune systems include: lymphoid: B cell, T cell (Cytotoxic Tcell, Natural Killer T cell, Regulatory T cell, T helper cell), Naturalkiller cell myeloid: granulocytes (Basophil granulocyte, Eosinophilgranulocyte, Neutrophil granulocyte/Hypersegmented neutrophil),Monocyte/Macrophage, Red blood cell (Reticulocyte), Mast cell,Thrombocyte/Megakaryocyte, Dendritic cell. The endocrine system includesthyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid(Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell),pineal (Pinealocyte) cells. Cells of the Nervous system are glial cells(Astrocyte, Microglia) Magnocellular neurosecretory cell, Stellate cell,Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope,Somatotrope, Lactotroph). Cells of the Respiratory system includePneumocyte (Type I pneumocyte, Type II pneumocyte), Clara cell, Gobletcell, Dust cell. Cells of the Circulatory system include Myocardiocyte,Pericyte. Cells of the Digestive system, Include stomach (Gastric chiefcell, Parietal cell), Goblet cell, Paneth cell, G cells, D cells, ECLcells, I cells, K cells, S cells. Enteroendocrine cells,Enterochromaffin cell, APUD cell, liver (Hepatocyte, Kupffer cell),Cartilage/bone/muscle. The integumentary system bone: Osteoblast,Osteocyte, Osteoclast, teeth (Cementoblast, Ameloblast) cartilage:Chondroblast, Chondrocyte skin/hair: Trichocyte, Keratinocyte,Melanocyte (Nevus cell), muscle: Myocyte, other: Adipocyte, Fibroblast,Tendon cell. Urinary system includes Podocyte, Juxtaglomerular cell,Intraglomerular mesangial cell/Extraglomerular mesangial cell, Kidneyproximal tubule brush border cell, Macula densa cell. The Reproductivesystem Includes male (Spermatozoon, Sertoli cell, Leydig cell), female(Ovum). Keratinizing epithelial cells include Epidermal keratinocyte(differentiating epidermal cell), Epidermal basal cell (stem cell),Keratinocyte of fingernails and toenails, Nail bed basal cell (stemcell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticularhair shaft cell, Cuticular hair root sheath cell, Hair root sheath cellof Huxley's layer, Hair root sheath cell of Henle's layer, External hairroot sheath cell, Hair matrix cell (stem cell), Wet stratified barrierepithelial cells, Surface epithelial cell of stratified squamousepithelium of cornea, tongue, oral cavity, esophagus, anal canal, distalurethra and vagina, basal cell (stem cell) of epithelia of cornea,tongue, oral cavity, esophagus, anal canal, distal urethra and vagina,Urinary epithelium cell (lining urinary bladder and urinary ducts),Exocrine secretory epithelial cells, Salivary gland mucous cell(polysaccharide-rich secretion), Salivary gland serous cell(glycoprotein enzyme-rich secretion), Von Ebner's gland cell in tongue(washes taste buds), Mammary gland cell (milk secretion), Lacrimal glandcell (tear secretion), Ceruminous gland cell in ear (wax secretion),Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweatgland clear cell (small molecule secretion). Apocrine sweat gland cell(odoriferous secretion, sex-hormone sensitive), Gland of Moll cell ineyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebumsecretion), Bowman's gland cell in nose (washes olfactory epithelium),Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminalvesicle cell (secretes seminal fluid components, including fructose forswimming sperm), Prostate gland cell (secretes seminal fluidcomponents), Bulbourethral gland cell (mucus secretion), Bartholin'sgland cell (vaginal lubricant secretion), Gland of Littre cell (mucussecretion), Uterus endometrium cell (carbohydrate secretion), Isolatedgoblet cell of respiratory and digestive tracts (mucus secretion),Stomach lining mucous cell (mucus secretion), Gastric gland zymogeniccell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloricacid secretion), Pancreatic acinar cell (bicarbonate and digestiveenzyme secretion), Paneth cell of small intestine (lysozyme secretion),Type II pneumocyte of lung (surfactant secretion), Clara cell of lung,Hormone secreting cells, Anterior pituitary cells, Somatotropes,Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediatepituitary cell, secreting melanocyte-stimulating hormone, Magnocellularneurosecretory cells, secreting oxytocin, secreting vasopressin, Gut andrespiratory tract cells, secreting serotonin. secreting endorphin,secreting somatostatin, secreting gastrin, secreting secretin, secretingcholecystokinin, secreting insulin, secreting glucagon, secretingbombesin, Thyroid gland cells, thyroid epithelial cell, parafollicularcell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell,Adrenal gland cells, chromaffin cells, secreting steroid hormones(mineralcorticoids and gluco corticoids), Leydig cell of testessecreting testosterone, Theca interna cell of ovarian follicle secretingestrogen, Corpus luteum cell of ruptured ovarian follicle secretingprogesterone, Granulosa lutein cells, Theca lutein cells,Juxtaglomerular cell (renin secretion), Macula densa cell of kidney,Metabolism and storage cells, Barrier function cells (Lung, Gut,Exocrine Glands and Urogenital Tract), Kidney, Type I pneumocyte (liningair space of lung), Pancreatic duct cell (centroacinar cell),Nonstriated duct cell (of sweat gland, salivary gland, mammary gland,etc.), Duct cell (of seminal vesicle, prostate gland, etc.), Epithelialcells lining closed internal body cavities, Ciliated cells withpropulsive function, Extracellular matrix secretion cells, Contractilecells. Skeletal muscle cells, stem cell, Heart muscle cells, Blood andimmune system cells, Erythrocyte (red blood cell), Megakaryocyte(platelet precursor), Monocyte, Connective tissue macrophage (varioustypes), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell(in lymphoid tissues), Microglial cell (in central nervous system),Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte,Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, NaturalKiller T cell, B cell, Natural killer cell, Reticulocyte, Stem cells andcommitted progenitors for the blood and immune system (various types),Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stemcells, adult stem cells, Nervous system, Sensory transducer cells,Autonomic neuron cells, Sense organ and peripheral neuron supportingcells, Central nervous system neurons and glial cells, Lens cells,Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germcells, Oogonium/Oocyte, Spermatid, Spermatocyte, Spermatogonium cell(stem cell for spermatocyte), Spermatozoon, Nurse cells, Ovarianfollicle cell, Sertoli cell (in testis), Thymus epithelial cell,Interstitial cells, or Interstitial kidney cells.

The cells can be healthy cells, or diseased cells. For example the cellscan be from a cancer condition such as epithelial cancer or carcinoma,including but not limited to, a, carcinoma of the prostate, carcinoma ofthe breast, carcinoma of the colon, pancreatic carcinoma, lungcarcinoma, skin carcinoma (melanoma), esophageal carcinoma, etc.) or theputative cell of origin (hepatocellular carcinoma, renal cell carcinoma,and small cell lung carcinoma, etc.). Other cancer cells includemyoepithelial cancers, sarcomas, gliomas, lymphomas, leukemias,carcinoids, and any other type of cancer. Cells in other states orconditions of tissue may be used including but not limited to,autoimmune conditions, immune system related conditions (e.g. allergies,likely immune response to challenge), cells representative of conditionsthat contribute to or exhibit resistance to standard treatments,susceptibility or predisposition to a condition (e.g. susceptibility todiabetes, thyroid conditions, stroke, cardiovascular conditions, orliver quality, function, and degeneration, etc.).

In some embodiments, the cell is a primary cell. In other embodiments,the cell is a mammalian cell. In some other embodiments, the cell is astem cell.

The cell surface can include any suitable native functional group, suchas amino acids and sugars. In some embodiments, the native functionalgroup can be an amino acid such as lysine, cysteine, tyrosine,threonine, serine, aspartic acid, glutamic acid or tryptophan. In otherembodiments, the native functional group is lysine. In some otherembodiments, the native functional group can be an N-terminal serine orthreonine.

The nucleic acid moiety can be any suitable nucleic acid moiety having anucleic acid or nucleotide. Exemplary nucleic acid moieties include, butare not limited to, an oligonucleotide, deoxy-ribonucleic acid (DNA),ribonucleic acid (RNA), peptide nucleic acid (PNA), Morpholino andlocked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleicacid (TNA), single-stranded DNA (ssDNA), aptamer, and others. Othernucleic acid moieties include fluorinated nucleic acids.

In another embodiment, the nucleic acid moieties of the invention arenucleic acids and also polymers of nucleotides. The term “nucleic acid”or “oligonucleotide” can be used interchangeably and are meant toinclude biopolymers of DNA or RNA nucleotides, including nucleic acidsthat are single-stranded, double stranded, triple stranded, branched orunbranched, or formed of a ladder of partially hybridizing shortoligonucleotides, nucleic acids having secondary structure, and/ornaturally occurring or non-naturally occurring nucleotides.

In one embodiment, a single-stranded oligonucleotide provides theopportunity to attach the cell by hybridization to its complementarystrand on another cell, a substrate surface, or a device.

The length of the single-stranded oligonucleotide used to attach on thecell surface can range from about 4 nucleotides to about 200nucleotides. Generally, a length of between about 12 nucleotides and 40nucleotides is optimal for hybridization. Strands of about 20 to about25 nucleotides are often used for hybridization purposes.

The number of nucleic acid moieties that are attached to the cellsurface can be upwards of about 100,000 per cell. In some embodiments,the number of nucleic acid moieties can be one nucleic acid moiety toabout 10,000, or about 30,000, or about 50,000. The number of nucleicacid moieties needed may vary based upon factors such as the applicationand/or cell type. In FIG. 1b , up to 120,000 DNA strands were shown tobe installed on each cell.

In one embodiment, the nucleic acid moiety is an aptamer. Aptamers areoligonucleic acid molecules that can adopt a three-dimensional structureand bind a specific target molecule. Aptamers are usually created byselecting them from a large random sequence pool, but natural aptamersalso exist in riboswitches. Aptamers can be used for both basic researchand clinical purposes as macromolecular drugs. Aptamers can be combinedwith ribozymes to self-cleave in the presence of their target molecule.These compound molecules have additional research, industrial andclinical applications. DNA or RNA aptamers are short strands of nucleicacid moieties. Aptamers are nucleic acid species that have beenengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. Aptamers are useful in biotechnological and therapeuticapplications as they offer molecular recognition properties that rivalthat of the commonly used biomolecule, antibodies. In addition to theirdiscriminate recognition, aptamers offer advantages over antibodies asthey can be engineered completely in a test tube, are readily producedby chemical synthesis, possess desirable storage properties, and elicitlittle or no immunogenicity in therapeutic applications. Aptamerselection includes a nucleic acid-based genetic regulatory elementcalled a riboswitch that possesses similar molecular recognitionproperties to the artificially made aptamers. This type of aptamer is anew mode of genetic regulation.

A concept of smart aptamers, and smart ligands discovers aptamers withpre-defined equilibrium (Kd), rate (koff, kon) constants andthermodynamic (ΔH, ΔS) parameters of aptamer-target interaction. Kineticcapillary electrophoresis selects the aptamers. Non-modified aptamersare cleared rapidly from the bloodstream, with a half-life of minutes tohours, mainly due to nuclease degradation and clearance from the body bythe kidneys, a result of the aptamer's inherently low molecular weight.Unmodified aptamer applications currently focus on treating transientconditions such as blood clotting, or treating organs such as the eyewhere local delivery is possible. This rapid clearance can be anadvantage in applications such as in vivo diagnostic imaging. An exampleis a tenascin-binding aptamer under development for cancer imaging.Several modifications, such as 2′-fluorine-substituted pyrimidines,polyethylene glycol (PEG) linkage, etc. (both of which are used inMacugen, an FDA-approved aptamer) are available to scientists with whichto increase the half-life of aptamers easily to the day or even weektime scale.

In addition to the development of aptamer-based therapeutics, manyresearchers have been developing diagnostic techniques for whole cellprotein profiling called proteomics, and medical diagnostics for thedistinction of disease versus healthy states. As a resource for all invitro selection the Aptamer Database catalogs all published experiments.This is found online at aptamer.icmb.utexas.edu/. AptaBiD orAptamer-Facilitated Biomarker Discovery is a technology for biomarkerdiscovery. AptaBiD is based on multi-round generation of aptamer or apool of aptamers for differential molecular targets on the cells whichfacilitates exponential detection of biomarkers. It involves three majorstages: (i) differential multi-round selection of aptamers for biomarkerof target cells; (ii) aptamer-based isolation of biomarkers from targetcells; and (iii) mass spectrometry identification of biomarkers. Theimportant feature of the AptaBiD technology is that it producessynthetic affinity probes (aptamers) simultaneously with biomarkerdiscovery. In AptaBiD, aptamers are developed for cell surfacebiomarkers in their native state and conformation. In addition tofacilitating biomarker identification, such aptamers can be directlyused for cell isolation, cell visualization, and tracking cells in vivo.They can also be used to modulate activities of cell receptors anddeliver different agents (e.g., siRNA and drugs) into the cells.

The affinities and selectivities of aptamers can rival those ofantibodies. In the present invention, the aptamers can be readilysite-specifically modified during chemical or enzymatic synthesis toincorporate particular reporters, linkers, or other moieties. Also,aptamer secondary structures can be engineered to undergoanalyte-dependent conformational changes, which, in concert with theability to specifically place chemical agents, allowing various possiblesignal transduction schemas, irrespective of whether the detectionmodality is optical, electrochemical, or mass based.

In another embodiment, the nucleic acid moiety is an oligonucleic acidsequence that can be used as an identifying sequence, a barcodesequence, a probe, a capture sequence for hybridization, a recognitionsequence, a gene expression control sequence, a gene sequence,enhancers, and/or sequences incorporating or derived fromnaturally-occurring enzymes, proteins, or other sequences.

In one embodiment, the nucleic acid moiety sequences attached to thecell are the same. In another embodiment, the nucleic acid moietysequences attached to the cell can be different. This would permit theattachment of nucleic acid moieties for multiples uses. For example, acapture nucleic acid moiety for the capture of the cell at a particularplacement, and hybridization or activated sequences to accomplish aspecific activity or utility.

In some embodiments, the nucleic acid moiety can be an oligonucleotide,DNA, RNA, PNA or an aptamer. In other embodiments, the nucleic acidmoiety can be single-stranded DNA (ssDNA). In some other embodiments,the nucleic acid moeity can be from about 10 to about 100 nucleic acids.In still other embodiments, the nucleic acid moiety can be an aptamer.

The nucleic acid moiety of the present invention can also include alinker. In another embodiment, a chemical linker for the cell attachmentsystem is used to link the oligonucleotide to the cell surface.Referring now to FIG. 1A, the linker facilitates binding to a cellmoiety on the cell surface such as an amino acid, carbohydrate, or othercell surface moiety. In one embodiment, the linker is a moiety that canbind directly to the amino acid on the cell without first modifying theamino acid (or carbohydrate or other moiety on the cell surface). Thechemical linker is placed at one end of the oligonucleotide to beattached. In one embodiment, formation of a bond with an amino acid onthe cell surface protein by the chemical linker alters its character anda covalent bond is formed between the amino acid and the nucleic acidoligonucleotide via the chemical linker. In some embodiments, the bondformed is an amide or ester bond. Thus, in the process of attaching tothe amino acid, the chemical linker will typically change its characterto form the amide, ester, or other bond, and the cell surface moietywill also conform to become part of the covalent bond.

In one specific embodiment, an N-hydrosuccinimide (NHS) ester is onesuch possible chemical linker, formed by the reaction of a carboxylatewith NHS in the presence of carbodiimide. NHS or sulfo-NHSester-containing reagents react with nucleophiles with release of theNHS or sulfo-NHS leaving group to form an acylated product. The reactionof such esters with a sulfhydryl or hydroxyl group forms ester linkagesor sulfohydryl ester linkages. Both of these bonds can potentiallyhydrolyze in aqueous environments or exchange with neighboring amines toform amide bonds and an NHS leaving group.

In another embodiment, the chemical linker is a heterobifunctionalcrosslinker. In one embodiment, the heterobifunctional crosslinker is aNHS-PEO_(n)-Maleimide. NHS-PEO_(n)-Maleimide reagents areheterobifunctional crosslinkers with N-hydroxysuccinimide (NHS) esterand maleimide groups that allow covalent conjugation of amine- andsulfhydryl-containing molecules.

In another embodiment, crosslinkers having polyethylene glycol (PEG),also referred to as polyethyleneoxide (PEO), spacers are convenientalternatives to reagents with purely hydrocarbon spacer arms. PEGspacers improve water solubility of reagent and conjugate, reduce thepotential for aggregation of the conjugate, and increases flexibility ofthe crosslink, resulting in reduced immunogenic response to the spaceritself. By contrast to typical PEG reagents that contain heterogeneousmixtures of different PEG chain lengths, these PEO reagents arehomogeneous compounds of defined molecular weight and spacer arm length,providing greater precision in optimization and characterization ofcrosslinking applications. For example,succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol] ester wasused in the examples to make a stock solution by dissolving 5 mg ofNHS-PEO₆-maleimide (Pierce Biotechnology, Inc. Rockford, Ill. 61105).

In another embodiment, the presence of sialic acid and EDC or1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (acarbomiide used for conjugating biological substances containingcarboxylates and amines), an NHS-maleimide can conjugate with asulfohydryl-oligonucleotide and reacted with an amino acid to form anester bond at the cell surface.

The amino acids possible for forming the amide, ester, or other bondwith the linker on the oligonucleotide include lysine, cysteine,aspartamate, glutamate, tyrosine, tryptophan and serine. Generally,lysine, cysteine, aspartamate, glutamate, and tyrosine form amide bondswith an NHS-oligonucleotide, and serine will form an ester bond with anNHS-oligonucleotide. Other linkers may form different bonds. For examplereagents including maleimide, disulfide and the process of acylation canbe used to form a direct covalent bond with a cysteine on a cell surfaceprotein. Amide coupling can be used at an aspartamate and glutamate toform an amide bond. Diazonium coupling, acylation, and alkylation can beused at a tyrosine on the cell surface to form an amide bond linkage. Itis possible that any of the amino acids (20 amino acids or any unnaturalamino acids) can be used to form the direct covalent bond that is theattachment of the oligonucleotide with the cell surface. The 20 aminoacids are isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, and valine (essential amino acids), and alanine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, and tyrosine, the nonessential amino acids, and also arginineand histidine.

In general, any affinity molecule useful in the prior art, incombination with a known ligand to provide specific recognition of adetectable substance will find utility in the attachment of nucleic acidgroups of the invention. Examples of such biological molecules which canthen be attached to these functional groups include linker moleculeshaving a known binding partner, or affinity molecule, include but arenot limited to, polysaccharides, lectins, selectins, nucleic acids (bothmonomeric and oligomeric), proteins, enzymes, lipids, antibodies, andsmall molecules such as sugars, peptides, aptamers, drugs, and ligands.

In another embodiment, the attachment is covalent. A bifunctionalcrosslinker useful for the invention would comprise two differentreactive groups capable of coupling to two different functional targetssuch as peptides, proteins, macromolecules, semiconductor nanocrystals,or substrate. The two reactive groups can be the same or different andinclude but are not limited to such reactive groups as thiol,carboxylate, carbonyl, amine, hydroxyl, aldehyde, ketone, activehydrogen, ester, sulfhydryl or photoreactive moieties. For examples, inone embodiment, a cross-linker can have one amine-reactive group and athiol-reactive group on the functional ends. Further examples ofheterobifunctional cross-linkers that may be used as linking agents inthe invention include but are not limited to:

-   -   amine reactive+sulfhydryl-reactive crosslinkers.    -   carbonyl-reactive+sulfhydryl-reactive crosslinkers.    -   amine-reactive+photoreactive crosslinkers    -   sulfhydryl-reactive+photoreactive crosslinkers    -   carbonyl-reactive+photoreactive crosslinkers.    -   carboxylate-reactive+photoreactive crosslinkers    -   arginine-reactive+photoreactive crosslinkers

Below is a list of categories in which crosslinkers generally fit. Thelist is exemplary and should not be considered exhaustive of the typesof crosslinkers that may be useful for the invention. For each category,i.e. which functional group these chemical target, there are somesubcategories, because one reactive group is capable of reacting withseveral functional groups.

Most crosslinkers with reactive groups can be broadly classified in thefollowing categories:

1. Amine-reactive: the cross-linker couple to a amine (NH2) containingmolecule.

2. Thiol-reactive: the cross-linker couple to a sulfhydryl (SH)containing molecule

3. Carboxylate-reactive: the cross-linker couple to a carboxylic acid(COOH) containing molecule.

4. Hydroxyl-reactive: the cross-linker couple to a hydroxyl (—OH)containing molecule.

5. Aldehyde- and ketone-reactive: the cross-linker couple to an aldehyde(—CHO) or ketone (R₂CO) containing molecule.

6. Active hydrogen-reactive.

7. Photo-reactive.

More specifically, chemical entering in these categories include, butare not limited to those containing:

-   -   1. Isothiocyanates, isocyanates, Acyl Azides, NHS esters,        Sulfonyl chlorides, aldehydes and glyoxals, epoxides and        oxiranes, carbonates, arylating agents, imidoesters,        carbodiimides, anhydrides, alkynes.    -   2. Haloacetyl and alkyl halide derivates, maleimides,        aziridines, acryloyl derivatives, arylating agents,        thiol-disulfides exchange reagents    -   3. Diazoalkanes and diazoacetyl compounds, such as        carbonyldiimidazoles and carbodiimides    -   4. Epoxides and oxiranes, carbonyldiimidazole, oxidation with        periodate, N,N′-disuccinimidyl carbonate or N-hydroxylsuccimidyl        chloroformate, enzymatic oxidation, alkyl halogens, isocyanates    -   5. Hydrazine derivatives for schiff base formation or reduction        amination    -   6. Diazonium derivatives for mannich condensation and iodination        reactions    -   7. Aryl azides and halogenated aryl azides, benzophenones, diazo        compounds, diazirine derivatives

For each of these subcategories there are many examples of chemicals.All these chemicals and the above list of subcategories are described inthe prior art, but many can be found in, “Bioconjugate Techniques” byGreg T Hermanson, Academic Press, San Diego, 1996, which is herebyincorporated by reference.

The choice of buffer solution wherein the conjugation and attachment tothe cells is carried out, depends on the choice of chemical linker orcrosslinker and maintaining growth conditions for cells (i.e., toprevent cell lysis). In a preferred embodiment, the buffer solutionrange is from pH 6-8 and should not contain the same functional groupsused in the chemical linker to react with the single-stranded nucleicacid. A pH of 7.2 is the median pH, but the pH does not have be neutral,but is dependent on compatibility with the chemical reaction and thecellular conditions.

In one embodiment, the buffer solution is a phosphate buffer solution ofneutral pH such that an N-hydrosuccinimide (NHS) ester (e.g.,NHS-PEO-maleimide) may be used as the chemical linker. The reaction isgenerally carried out under conditions that allow the conjugation of thechemical linker and the nucleic acid and the subsequent attachment tothe cell or cell surface. In some embodiments where an NHS estercrosslinker and phosphate buffer solution is used, the reactions arecarried out at neutral pH (e.g., pH 7.2) and at room temperature for aspecified period of time (e.g., 1, 3, 5, 10, 15, 20, 30, 45, 60 or moreminutes).

In some embodiments, the conjugate includes a mammalian cell havinglysine on the cell surface, and a single-stranded DNA covalently linkedto the lysine via an amide.

The conjugates of the present invention can be prepared by any suitablemeans known in the art. In general, the method involves linking DNA tothe native functional group of a cell where the cell is unmodified, butisolating the cell from other biological material that might interferewith the conjugation step. Any bioconjugation technique can be used,such as those described above in Hermanson.

In some embodiments, the present invention includes a method ofpreparing a conjugate of a cell and a nucleic acid moiety, by contactingthe cell with an activated nucleic acid moiety, wherein the cell has asurface including a native functional group and wherein the cell has nocell wall, such that the nucleic acid moiety is covalently linked to thenative functional group.

The activated nucleic acid moiety includes a linker, as described above.In some embodiments, the activated nucleic acid moiety includes anactivated ester. In other embodiments, the method of making theconjugates includes contacting a mammalian cell with an activatednucleic acid moiety, wherein the native functional group includes lysineand the activated nucleic acid moiety includes an NHS-ester, such thatthe nucleic acid moiety is covalently linked to the native functionalgroup by amide bond formation.

B. Conjugates of Cells with Cell Walls

The present invention also provides conjugates of nucleic acid moietiesand cells with cell walls, as well as methods of making. In someembodiments, the present invention provides a conjugate of a cell havinga cell wall, and a nucleic acid moiety wherein the nucleic acid moietyis covalently linked to the cell. In other embodiments, the nucleic acidmoiety is covalently linked to the cell surface.

The native functional group can be any suitable native functional group,such as an amino acid or sugar. Sugars useful for linking to the nucleicacid moiety include, but are not limited to, glucose, ribose, fructose,sialic acid, mannose, galactose, sucrose, lactose, and others. Othersugars include, but are not limited to monosaccharides, disaccharidesand polysaccharides.

In another embodiment, attachment of oligonucleotides to cell surfacescan be accomplished with plant cells, bacterial cells, fungi, yeast,algae, and archaea. The attachment in this case requiring first amodification of a carbohydrate molecule on the cell surface, and thenattachment of an oligonucleotide-linker to the modified moiety. Themajor difference between animal cells and plant cells is that the plantcells have cell walls, thus requiring modification before attachment ofthe oligonucleotide. In one embodiment, attachment of oligonucleotidescan be achieved through periodate oxidation followed by hydrazoneformation. In one embodiment, a carbohydrate on the plant cell can bemodified instead of modifying a protein as in other embodiments usingmammalian or animal cells. In the present example, the carbohydratesugar is oxidized to generate a functional aldehyde (or a ketone). Analdehyde or ketone is then reacted with synthetic hydrizido-DNA to forma covalent bond called a hydrazone.

More generally, the composition comprises an oligonucleotide conjugatedto a chemical linker, the linker covalently attached to an outsidesurface of the cell. The bond or linkage is generally a hydrazone, anoxime, or an amine. The method to form the bond between theoligonucleotide and the cell surface carbohydrate includes modifying thenon-animal cell surface carbohydrate to form an aldehyde or a ketone.The process of this formation can include periodate oxidation. Thealdehyde or ketone is then contacted with an oligonucleotide having afunctional group that reacts to form a covalent bond. The covalent bondcan be a hydrazone, an oxime, or an amine linkage between the linker onthe oligonucleotide and the carbohydrate on the surface of the cell.Generally, the linkage with these bonds is at a carbohydrate on the cellsurface. The reaction can be performed with a non-animal cell or ananimal cell, preferably with non-animal cells such as plants, yeast,bacteria and algae and other unicellular organisms. In some embodiments,the cell is a plant cell.

In other embodiments, the native functional group includes a modifiednative functional group. As described above, the native functional groupcan be a sugar having a 1,2-diol group that is oxidized to formaldehydes or ketones. In some other embodiments, the modified nativefunctional group includes an oxidized sugar. In still other embodiments,the modified native functional group includes a sialic acid, mannose,glucose, galactose, N-acetylglucosamine or N-acetylmannosamine.

The present invention also provides a method of making the conjugates ofa nucleic acid moiety and a cell with a cell wall. As described above,the method can include a two-step process of first modifying the nativefunctional group of the cell having a cell wall, followed by conjugationof the nucleic acid moiety to the modified native functional group.

In some embodiments, the present invention provides a method forpreparing a conjugate of a cell and a nucleic acid moiety, includingcontacting the cell with an activated nucleic acid moiety wherein thecell has a cell wall, such that the nucleic acid moiety is linked to thecell. The activated nucleic acid moiety can include any suitableactivated group to enable conjugation of the nucleic acid moiety to thecell, as described above. In some embodiments, the activated nucleicacid moiety includes an aminooxy, a hydrazide, a hydrazine, asemicarbazide, a thiosemicarbazide or an amine. Alternatively, theactivated nucleic acid moiety can include cysteine to form thiazolidinesor serine to form oxazolidines. Other methods of conjugating the nucleicacid moiety and cell are described in Hermanson (see above).

The method of preparing the conjugate with a cell having cell walls alsoincludes the step of modifying the native functional group. In someembodiments, the method of preparing the conjugate of a nucleic acidmoiety and a cell with cell walls, includes contacting the nativefunctional group with a modifying agent to prepare a modified nativefunctional group, such that the nucleic acid moiety is covalently linkedto the modified native functional group.

The modifying agent can be any suitable agent to prepare the modifiednative functional group. For example, the modifying agent includes, butis not limited to, an oxidizing agent. In some embodiments, themodifying agent includes an oxidizing agent. Suitable oxidizing agentsinclude, but are not limited to, sodium periodate. In other embodiments,the oxidizing agent includes sodium periodate. In some otherembodiments, the modified native functional group includes an oxidizedsugar. In still other embodiments, the modified native functional groupincludes an oxidized sialic acid. In yet other embodiments, the modifiednative functional group includes an aldehyde group.

IV. Devices

The present invention also provides substrate surfaces and devices thatinclude the conjugates of the present invention. In some embodiments,the present invention provides a device including a cell having a cellsurface including a native functional group covalently linked to a firstnucleic acid moiety. The device also includes a substrate surfaceincluding a second nucleic acid moiety complementary to the firstnucleic acid moiety, such that the cell is bound to the substratesurface via formation of a nucleic acid duplex of the first and secondnucleic acid moieties.

In some embodiments, the cell is an animal cell.

The substrate surface can be of any suitable material. Examples ofsuitable materials include, but are not limited to, glass (includingcontrolled-pore glass), polymers (e.g., polystyrene, polyurethane,polystyrene-divinylbenzene copolymer), silicone rubber, quartz, latex, aderivatizable transition metal, magnetic materials, silicon dioxide,silicon nitride, gallium arsenide, and derivatives thereof. Thesubstrate surface can also have any suitable surface geometry,including, but not limited to, planar, curved and spherical.

In some embodiments, the substrate surface is planar. In otherembodiments, the substrate surface is spherical.

In some embodiments, the device also includes channels for fluid. Inother embodiments, the channels are microchannels. In some otherembodiments, the channels are nanochannels.

In some embodiments, the device includes a sensor. In other embodiments,the sensor includes a nanosensor. In some other embodiments, the sensorincludes an electrode. In still other embodiments, the sensor includes apiezoelectric sensor. In yet other embodiments, the device is adaptedfor atomic force microscopy.

In some embodiments, the device is adapted for biochemical orelectrochemical analysis of the cell. In other embodiments, thebiochemical analysis includes genomic analysis. In some otherembodiments, the device also includes a component selected from amicrofabricated heater, a temperature sensor, polymerase chain reaction(PCR) chambers or capillary electrophoretic separation channels. Instill other embodiments, the device is capable of generating atranscriptional profile of the cell.

In some embodiments, the device also includes a bioreactor.

In another embodiment, the present invention provides a platform for useto observe and screen for processes that can be studied through cellcapture by oligonucleotide attachment such as wound healing, tissueregeneration, infection, reactivity or responsiveness to test drugs,drug screening generally, and gene expression in response to stimuli.The present invention also provides methods and synthetic systems thatpermit various human states and conditions (e.g., diseased and normal)to be studied or detected through cell capture by oligonucleotideattachment. For example, oligonucleotide attachment to cells may also beapplied in the practice of diagnosis of a disease by detection of a cellsurface marker of the disease by detecting the hybridization of the cellsurface marker to a capture oligonucleotide.

Cells having oligonucleotide strands attached on their surface can behybridized or annealed to each other in three dimensions, such as fluidor gel. Many applications of this technology are possible. Athree-dimensional scaffold or mesh can be used to pattern many morecells than can be displayed on a planar surface. Thus, in oneembodiment, a mesh, porous bulk material, or scaffold can be modified todisplay oligonucleotides attached to the surface such that cellsmodified using the present methods can be displayed or captured on thesurface. Such surfaces can be used to study genetics, aging, and drugresponse or for metabolic engineering and production and collection ofcellular byproducts.

For example the cells can be used to study tissue regeneration, such asmyocardial tissue regeneration, wherein a critical number of cells forma beating unit upon accumulation of the sufficient number of cells. Anytissue can potentially be “grown” using a seeded cell attachment matrixto generate a network of interacting cells. The dynamics and products ofsuch cell aggregates and simulated tissues can be studied andcontrolled. For example, stem cell differentiation and storage can befacilitated using the cell attachment system. For example, adult stemcells and induced pluripotent stem cells can be kept fromdifferentiation or guided to a differentiated state as a desired celltype. Artificial tissues can be generated for replacing lost, damaged,or diseased tissue in animals including humans. Neural and spinal tissueregeneration can be effected in the proper environment in vitro or invivo. In order to guide or study the cells in such a system, a sensormay be positioned within the cells or on a surface in contact with thecells.

The cell attachment system can be used for delivery purpose, using thecell with attached oligonucleotides to deliver other cell surfacemolecules such as aptamers, gene regulating molecules, gene expressioncontrol molecules, genes for control of gene expression. The cellattachment system can also be used to deliver to another cell thecontents of the cell having the oligonucleotides attached. For example,small molecules, peptides, peptidomimetics, vectors having DNA for geneexpression, interfering RNA molecules such as small inhibitor RNA orshort hairpin RNA, and other deliverable molecules. An aptamer on thesurface of the cell can bind an internalizing receptor and the cell isinternalized with its contents. Generally, a cell having on its surfacean oligonucleotide that is an aptamer adapted to bind a protein willprovide the delivery mechanism to or into another living cell. Theaptamer itself may comprise a nucleic acid adapted to act within thecell such as an oligonucleotide for expression in the cell, aninterfering RNA, (such as an siRNA, an shRNA, or a microRNA) or amolecular beacon such as a nucleic acid having a label adapted to bind aspecific sequence in the cell and provide optical detection of thatsequence or gene represented by that sequence. The nucleic acids canenhance gene expression, control gene expression, block gene expression,or modify gene expression, among other genomic modifying activities.

In another embodiment, the invention provides methods for sequencespecific patterning or capture of cells on a substrate and screeningmethods. For example, a cell or cell sample can be incubated with PBSphosphate buffer pH 7.2 and the linker for several minutes to severalhours as described in the examples to attach the single-strandedoligonucleotide to the cell surface, thus resulting in a cell modifiedon its surface with an oligonucleotide. A substrate surface can beprepared having a sequence (e.g., a ssDNA capture sequence) attached tothe substrate surface. In one embodiment, the single-strandedoligonucleotide attached to the cell is complementary to the ssDNAcapture sequence attached to the substrate such that when the cell isallowed to contact the substrate, the two sequences hybridize, thusimmobilizing the cell to the substrate.

Thus, in another embodiment, the present invention further providesdevices featuring cells immobilized on a surface using the presentmethods of cell modification. In one embodiment, the devices of thisinvention can be sensors patterned in microarrays or microarray-likepatterns. In another embodiment, the cell or cell membrane witholigonucleotides attached can be used in a device for analyzing the cellor other purposes. In some embodiments, the device will havecomplementary oligonucleotides on its surface for hybridizing to anoligonucleotide attached to a cell using the present methods. The devicecan be any useful shape, for example planar or spherical. It can havechannels for fluid (i.e., micro channels or nanochannels). The devicecan include a sensor, such as a nanosensor, an electrode, or a piezoelectrode. The device can be adapted for atomic force microscopy,biochemical analysis of the cell, or genomic analysis. The device canhave additional components such as a heater, a temperature sensor, andcomponents such as PCR chambers and capillary electrophoretic separationchannels for generating a transcriptional profile of the cell, amongother possible processes. The device can further comprise a bioreactor.The device can comprise a component selected from a microfabricatedheater, a temperature sensor, polymerase chain reaction (PCR) chambersand capillary electrophoretic separation channels.

In one embodiment, for detection of the hybridization event theoligonucleotide attached to the cell or the complementaryoligonucleotide or capture oligonucleotide on the device is labeleddepending on the device. For example, if the label is added with theamplification mix, the cDNA is on the template strand while the probesare on the sense strand (unless they are negative controls). The labelis typically fluorescent, although occasionally radiolabels and the likeare used. The labeling can be direct or indirect. Indirect labelingrequires a coupling stage which can occur before or after hybridization.If labeling occurs before hybridization, hybridization (e.g., intwo-channel arrays) nucleotides labeled with dyes such as aminoallyl-UTPand NHS amino-reactive dyes (like cyanine dyes) can be employed. Theaminoallyl group is an amine group typically on a long linker attachedto the nucleobase, which reacts with a reactive dye. In someembodiments, the modified nucleotides (typically a 1 aaUTP: 4 TTP mix)are added enzymatically at a lower rate compared to normal nucleotides,typically resulting in 1 every 60 bases as measured with aspectrophotometer. The aaDNA is then purified with a column for example,using solution containing Tris phosphate buffer containing amine groups.

Microarrays can be fabricated using a variety of technologies, includingprinting with fine-pointed pins onto glass slides, photolithographyusing pre-made masks, photolithography using dynamic micromirrordevices, ink-jet printing, or electrochemistry on microelectrode arrays.In spotted microarrays, the probes are oligonucleotides, cDNA or smallfragments of PCR products that correspond to mRNAs. The probes aresynthesized prior to deposition on the array surface and are then“spotted” onto glass. A common approach utilizes an array of fine pinsor needles controlled by a robotic arm that is dipped into wellscontaining DNA probes and then depositing each probe at designatedlocations on the array surface. The resulting “grid” of probesrepresents the nucleic acid profiles of the prepared probes and is readyto receive complementary cDNA or cRNA “targets” derived fromexperimental or clinical samples.

In one embodiment, the oligonucleotide probes in these oligonucleotidemicroarrays are short sequences designed to match parts of the sequenceof known or predicted open reading frames. In such an array, theoligonucleotide arrayed on the substrate can be produced by printingshort oligonucleotide sequences designed to represent a single gene orfamily of gene splice-variants by synthesizing this sequence directlyonto the array surface instead of depositing intact sequences. Sequencesmay be longer (60-mer probes such as the Agilent design) or shorter(25-mer probes produced by Affymetrix) depending on the desired purpose;longer probes are more specific to individual target genes, shorterprobes may be spotted in higher density across the array and are cheaperto manufacture. One technique used to produce oligonucleotide arraysinclude photolithographic synthesis (Agilent and Affymetrix) on a silicasubstrate where light and light-sensitive masking agents are used to“build” a sequence one nucleotide at a time across the entire array.Each applicable probe is selectively “unmasked” prior to bathing thearray in a solution of a single nucleotide, then a masking reactiontakes place and the next set of probes are unmasked in preparation for adifferent nucleotide exposure. After many repetitions, the sequences ofevery probe become fully constructed. More recently, Maskless ArraySynthesis from NimbleGen Systems has combined flexibility with largenumbers of probes.

Two-color microarrays or two-channel microarrays are typicallyhybridized with cDNA prepared from two samples to be compared (e.g.diseased tissue versus healthy tissue or diseased cells versus healthycells) and that are labeled with two different fluorophores. Fluorescentdyes commonly used for cDNA labelling include Cy3, which has afluorescence emission wavelength of 570 nm (corresponding to the greenpart of the light spectrum), and Cy5 with a fluorescence emissionwavelength of 670 nm (corresponding to the red part of the lightspectrum). The two Cy-labelled cDNA samples are mixed and hybridized toa single microarray that is then scanned in a microarray scanner tovisualize fluorescence of the two fluorophores after excitation with alaser beam of a defined wavelength. Relative intensities of eachfluorophore may then be used in ratio-based analysis to identifyup-regulated and down-regulated genes.

The present cell modifications can be made to cells and used inmicrofluidics applications, methods and devices. The present methods ofcell surface modification enable the capture and immobilization of asingle cell onto a surface, which thereby permits the cell to be actedon in various ways. For example, in a specific embodiment, an integratedmicrofluidic device can be made such as that described in Example 4 anddescribed in Toriello, et al, Integrated microfluidic bioprocessor forsingle-cell gene expression analysis, Proc. Natl. Acad. Sci. U.S.A.,105, 20173-20178, 2008 December 23; 105(51):20173-8. Epub 2008 December15, hereby incorporated by reference. This integrated microdevice wasdeveloped for the analysis of gene expression in single cells.Reverse-transcription PCR amplification of a single cell is enabled bythe immobilization and capture of a single-cell on the surface of thisintegrated microdevice.

Thus in one embodiment, the present methods enable microfluidicstructures which feature immobilized cells using the present direct cellmodifications. Such microfluidic structures may include micropneumaticsystems, i.e. microsystems for the handling of off-chip fluids (liquidpumps, gas valves, etc), and microfluidic structures for the on-chiphandling of nano- and picoliter volumes for use in various molecularbiology procedures, such as for enzymatic analysis (e.g., glucose andlactate assays), DNA analysis (e.g., polymerase chain reaction andhigh-throughput sequencing), and proteomics.

Such microfluidic devices are intended to integrate assay operationssuch as detection, as well as sample pre-treatment and samplepreparation on one chip. An emerging application area for biochips isclinical pathology, especially the immediate point-of-care diagnosis ofdiseases. In addition, microfluidics-based devices, capable ofcontinuous sampling and real-time testing of air/water samples forbiochemical toxins and other dangerous pathogens, can serve as abiosensor (e.g., a “bio-smoke alarm”) for early warning.

In another embodiment, microfluidics for use with cells havingoligonucleotides attached by the present method include continuous-flowtechnologies based on the manipulation of continuous liquid flow throughmicrofabricated channels. Actuation of liquid flow is implemented eitherby external pressure sources, external mechanical pumps, integratedmechanical micropumps, or by electrokinetic mechanisms. Continuous-flowmicrofluidic operation is the mainstream approach because it is easy toimplement and less sensitive to protein fouling problems.Continuous-flow devices are adequate for many well-defined and simplebiochemical applications, and for certain tasks such as chemicalseparation, but they are less suitable for tasks requiring a high degreeof flexibility or complicated fluid manipulations. Process monitoringcapabilities in continuous-flow systems can be achieved with highlysensitive microfluidic flow sensors based on MEMS technology which offerresolutions down to the nanoliter range.

Other embodiments include but are not limited to, Digital(droplet-based) microfluidic alternatives to the above closed-channelcontinuous-flow systems including novel open structures, where discrete,independently controllable droplets are manipulated on a substrate.

In addition to microarrays, another embodiment comprises biochipsdesigned for two-dimensional electrophoresis, transcriptome analysis,PCR amplification, etc. Other applications include variouselectrophoresis and liquid chromatography applications for proteins andDNA, cell separation, in particular blood cell separation, proteinanalysis, cell manipulation and analysis including cell viabilityanalysis and microorganism capturing.

In another embodiment, the present direct cell modification methodsenable a device for single cell imaging, manipulation and patterning.For example, cells can be analyzed or patterned using an atomic forcemicroscope (AFM) or scanning force microscope (SFM). Thus, in oneembodiment, a device such as an AFM cantilever as described in Hsiao S Cet al., DNA-coated AFM cantilevers for the investigation of celladhesion and the patterning of live cells, Angew Chem Int Ed Engl. 2008;47(44):8473-7, 16 Sep. 2008 Epub, hereby incorporated by reference, andalso described in Example 8. The advantages the technology embodied inthe invention bring to an AFM device is an ability to precisely placecells using AFM cantilevers for analysis and manipulation. Both AFM andSFM are very high-resolution type of scanning probe microscopes, withdemonstrated resolution of fractions of a nanometer, more than 1000times better than the optical diffraction limit.

The ability to subject a whole live cell to an AFM using theoligonucleotide attachment system of the invention provides theopportunity to specifically analyze multiple cell types, simultaneously,and indeed to analyze a single cell with greater fidelity because it issecurely held and controlled from its oligonucleotide anchors. Forexample, another major application of AFM (besides imaging) is forcespectroscopy, the measurement of force-distance curves. For this method,the AFM tip is extended towards and retracted from the surface as thestatic deflection of the cantilever is monitored as a function ofpiezoelectric displacement. These measurements have been used to measurenanoscale contacts, atomic bonding, Van der Waals forces, and Casimirforces, dissolution forces in liquids and single molecule stretching andrupture forces. Forces of the order of a few pico-Newtons can now beroutinely measured with a vertical distance resolution of better than0.1 nanometer.

Micro and nanotechnology are being applied to chemical analysis,environmental monitoring, medical diagnostics and cellomics andmicroreactors for pharmaceutics. Research in lab on a chip (LOC) systemsis expected to extend towards downscaling of fluid handling structuresas well, by using nanotechnology. Sub-micrometre and nano-sizedchannels, DNA labyrinths, single cell detection an analysis andnano-sensors might become feasible that allow new ways of interactionwith biological species and large molecules. The present inventioncontributes to making these systems possible for whole cells. LOCsystems can accomplish real-time PCR, facilitate biochemical assays,immunoassay, detect bacteria, viruses and cancers based onantigen-antibody reactions, dielectrophoresis detecting cancer cells andbacteria, blood sample preparation, can crack cells to extract DNA,cellular analysis, on channel screening.

Lab-on-a-chip technology may soon become an important part of efforts toimprove global health, particularly through the development ofpoint-of-care testing devices. In countries with few healthcareresources, infectious diseases that would be treatable in a developednation are often deadly. In some cases, poor healthcare clinics have thedrugs to treat a certain illness but lack the diagnostic tools toidentify patients who should receive the drugs. Many researchers believethat LOC technology may be the key to powerful new diagnosticinstruments. The goal of these researchers is to create microfluidicchips that will allow healthcare providers in poorly equipped clinics toperform diagnostic tests such as immunoassays and nucleic acid assayswith no laboratory support. An innovative polymer lab-on-a-chip (LOC)for reverse transcription (RT)-polymerase chain reaction (PCR) has beendesigned, fabricated, and characterized for point-of-care testing (POCT)clinical diagnostics. In addition, a portable analyzer that consists ofa non-contact infrared (IR) based temperature control system for RT-PCRprocess and an optical detection system for on-chip detection, has alsobeen developed and used to monitor the RT-PCR LOC.

In another embodiment, a fully integrated genomic analysis microsystemincluding microfabricated heaters, temperature sensors, and PCR chambersdirectly connected to capillary electrophoretic separation channels hasbeen constructed. In Example 6, an integrated microfluidic bioprocessorfor single-cell gene expression analysis is described and also describedin Toriello et al., “Integrated microfluidic bioprocessor forsingle-cell gene expression analysis”, PNAS, Dec. 23, 2008 vol. 105 no.51 20173-20178, online on 9/16/08, and hereby incorporated by reference.The device is an important step toward a microfabricated genomicmicroprocessor for use in forensics and point-of-care molecular medicaldiagnostics. Whether the cells are displayed on a device, or connectedto one another, analysis of gene expression can be determined bymeasuring mRNA levels with multiple techniques including microarrays,expressed cDNA sequence tag (EST) sequencing, serial analysis of geneexpression (SAGE) tag sequencing, massively parallel signaturesequencing (MPSS), or various applications of multiplexed in-situhybridization. All of these techniques are extremely noise-prone and/orsubject to bias in the biological measurement, and a major research areain computational biology involves developing statistical tools toseparate signal from noise in high-throughput gene expression studies.Such studies are often used to determine the genes implicated in adisorder: one might compare microarray data from cancerous epithelialcells to data from non-cancerous cells to determine the transcripts thatare up-regulated and down-regulated in a particular population of cancercells.

Analysis of regulation can likewise occur in these oligonucleotidelinked cells: regulation is the complex orchestration of events startingwith an extracellular signal such as a hormone and leading to anincrease or decrease in the activity of one or more proteins.Bioinformatics techniques have been applied to explore various steps inthis process. For example, promoter analysis involves the identificationand study of sequence motifs in the DNA surrounding the coding region ofa gene. These motifs influence the extent to which that region istranscribed into mRNA. Expression data can be used to infer generegulation: one might compare microarray data from a wide variety ofstates of an organism to form hypotheses about the genes involved ineach state. In a single-cell organism, one might compare stages of thecell cycle, along with various stress conditions (heat shock,starvation, etc.). One can then apply clustering algorithms to thatexpression data to determine which genes are co-expressed. For example,the upstream regions (promoters) of co-expressed genes can be searchedfor over-represented regulatory elements.

In another embodiment, the presently modified cells with anoligonucleotide attached is used in or integrated into a sensor devicethat measures a physical quantity and converts it into a signal whichcan be read by an observer or by an instrument. For example, analysis ofprotein expression can also be accomplished with these compositions andsystems. Protein microarrays and high throughput (HT) mass spectrometry(MS) can provide a snapshot of the proteins present in a cell.Bioinformatics is very much involved in making sense of proteinmicroarray and HT MS data; the former approach faces similar problems aswith microarrays targeted at mRNA, the latter involves the problem ofmatching large amounts of mass data against predicted masses fromprotein sequence databases, and the complicated statistical analysis ofsamples where multiple, but incomplete peptides from each protein aredetected.

Analysis of mutations in cancer, or any other disease or conditions canbe accomplished by these systems using the oligonucleotide capturedcells. In cancer, the genomes of affected cells are rearranged incomplex or even unpredictable ways. Massive sequencing efforts are usedto identify previously unknown point mutations in a variety of genes incancer. Bioinformaticians continue to produce specialized automatedsystems to manage the sheer volume of sequence data produced, and theycreate new algorithms and software to compare the sequencing results tothe growing collection of human genome sequences and germlinepolymorphisms. New physical detection technology are employed, such asoligonucleotide microarrays to identify chromosomal gains and losses(called comparative genomic hybridization), and single nucleotidepolymorphism arrays to detect known point mutations. These detectionmethods simultaneously measure several hundred thousand sites throughoutthe genome, and when used in high-throughput to measure thousands ofsamples, generate terabytes of data per experiment. Again the massiveamounts and new types of data generate new opportunities forbioinformaticians. The data is often found to contain considerablevariability, or noise, and thus Hidden Markov model and change-pointanalysis methods are being developed to infer real copy number changes.In the structural branch of bioinformatics, homology is used todetermine which parts of a protein are important in structure formationand interaction with other proteins. In a technique called homologymodeling, this information is used to predict the structure of a proteinonce the structure of a homologous protein is known. This currentlyremains the only way to predict protein structures reliably.

Comparative genomics can be made with the systems of the invention. Thecore of comparative genome analysis is the establishment of thecorrespondence between genes (orthology analysis) or other genomicfeatures in different organisms. It is these intergenomic maps that makeit possible to trace the evolutionary processes responsible for thedivergence of two genomes. A multitude of evolutionary events acting atvarious organizational levels shape genome evolution. At the lowestlevel, point mutations affect individual nucleotides. At a higher level,large chromosomal segments undergo duplication, lateral transfer,inversion, transposition, deletion and insertion. Ultimately, wholegenomes are involved in processes of hybridization, polyploidization andendosymbiosis, often leading to rapid speciation. The complexity ofgenome evolution poses many exciting challenges to developers ofmathematical models and algorithms, who have recourse to a spectra ofalgorithmic, statistical and mathematical techniques, ranging fromexact, heuristics, fixed parameter and approximation algorithms forproblems based on parsimony models to Markov Chain Monte Carloalgorithms for Bayesian analysis of problems based on probabilisticmodels.

The cells and systems can be used for making prognoses and diagnoses ofpatients. Individual genomes can be genotyped and analyzed using thedevices enabled by direct cell modification. For example, a genotypingstage can have many different experimental approaches including singlenucleotide polymorphism (SNP) chips (typically 0.02% of the genome), orpartial or full genome sequencing. Once the genotypes are known, thereare many bioinformatics analysis tools that can compare individualgenomes and find disease association of the genes and loci.

In another embodiment, the presently modified cells are used in abioreactor device or system to facilitate bioactivity in a cell orbetween several molecules, and generally resulting in a finished productor some other desired result such as a bioactivity (e.g. metabolism,energy production, electrical signals, catabolism, apoptosis, growth,differentiation, proliferation, etc.). For example, metabolism of a cellcan be studied in these systems. A bioreactor may refer to any device orsystem that supports a biologically active environment.

Scientific advances in biomaterials, stem cells, growth anddifferentiation factors, and biomimetic environments have created uniqueopportunities to fabricate tissues in the laboratory from combinationsof engineered extracellular matrices (“scaffolds”), cells, andbiologically active molecules. In another embodiment, the present cellmodification methods enable cells to be attached or placed on othersurfaces meant to grow cells or tissues in the context of cell culture.For example, cells having single stranded oligonucleotides attached by acovalent bond to their surface can be hybridized to a complementarystrand on a neighboring cell or on a surface, therefore providing themeans for linking cells together and/or to a surface for generation andstudy of cells, tissues, and organs. Powerful developments in themultidisciplinary field of tissue engineering have yielded a novel setof tissue replacement parts and implementation strategies which thepresent methods will find use. In one embodiment, cells modified with anoligonucleotide attached can be implanted or ‘seeded’ into an artificialstructure capable of supporting three-dimensional tissue formation. Bulkmaterials, stents, scaffolds, which are often critical, both ex vivo aswell as in vivo, to recapitulating the in vivo milieu and allowing cellsto influence their own microenvironments can be modified to allow cellattachment using the present methods.

In one embodiment, a photosynthetic single-celled organism is modifiedusing the present methods and an oligonucleotide is attached. Proposedorganisms include but are not limited to, Photosynthetic: C. reinhardtii(algae), Synechocystis PCC 6803 (cyanobacteria), R. rubrum (gramnegative anaerobe); Heterotrophs: C. pasteurianum (nitrogen fixer),Azotobacter sp. (proposed to have the highest respiration rate of anyorganism). (see Melis et al, cited elsewhere herein).

In one embodiment, algae is modified and an oligonucleotide is attached.Algae may be especially suitable because it can grow rapidly and canhave a high percentage of lipids, or oils. They can double their massseveral times a day and produce at least 15 times more oil per acre thanalternatives such as rapeseed, palms, soybeans, or jatropha. Due to itslack of need for clean water, algae farming is also more cost effectivethan many other crops, and produces much less strain on fresh waterresources. It can also be grown without displacing food crops. Theoligonucleotide attachment system allows control of the placement of aplant, bacterial, or algae cell on a surface, for example, a surfaceangled to maximize exposure to sunlight.

In another embodiment, the presently modified cells with anoligonucleotide attached can be used to in a fuel cell or otherelectrochemical conversion device. It produces electricity from fuel (onthe anode side) and an oxidant (on the cathode side), which react in thepresence of an electrolyte. The reactants flow into the cell, and thereaction products flow out of it, while the electrolyte remains withinit. Fuel cells can operate virtually continuously as long as thenecessary flows are maintained. Fuel cells are different fromelectrochemical cell batteries in that they consume reactant from anexternal source, which must be replenished—a thermodynamically opensystem. By contrast batteries store electrical energy chemically andhence represent a thermodynamically closed system. Many combinations offuel and oxidant are possible. A hydrogen cell uses hydrogen as fuel andoxygen (usually from air) as oxidant. Other fuels include hydrocarbonsand alcohols. Other oxidants include chlorine and chlorine dioxide.

Living solar cells can be created using the green algae Chlamydomonasreinhardtii for microbial electricity generation as described inRosenbaum, Appl. Microbiol. Biotechnol. (2005) 68: 753-756. Twochambered microbial fuel cells are described in Wang et al,Electrochimica Acta 54 (2009) 1109-1114. Self-sustained phototrophicmicrobial fuel cells (MFCs) are described in He et al, Environ. Sci.Technol. 2009, 43, 1648-1654 in which production of electricity fromself-sustained sediment phototrophic MFC was achieved using a mixedmicrobial community of photosynthetic microorganisms and heterotrophicbacteria. Electricity was constantly generated from these MFCs with noinput of organic compounds or nutrients. Kjeang et al (Journal of PowerSources 158 (2006) 1-12) describe strategic enzyme patterning formicrobial fuel cells to optimize a combined fuel and oxidant channels ina non-compartmentalized fuel cell assembly with separated enzymespatterned in the device in relation to individual turnover rates.Ringeisen et al. J. of Power Sources 165(2007) 591-597 describes aminiature MFC constructed using Shewanella (DSP10) oneidensis thatremains active in anaerobic and aerobic environments. Previous studiesshowed that electrons from this bacteria have been used to reduce metalsin the presence of oxygen. The bacteria was used in the devices as theactived electrochemical species in the anode chamber. The paper pointsout that sensors for underwater surveillance systems require as an idealpower source a small (stealthy, not dominating in size compared to thedevice it powers) that can function in an aerobic environment (ie. in awater column close to the water surface), providing continuous power(with no recharging or lifetime issues) and not requiring high levels ofsolar radiation (therefore functioning subsurface and at night time,etc.). The bacteria selected for the study by this group provides theopportunity for RF communication for a sensor network because it doesnot have to be in an anaerobic environment for the anode, and thereforecan function in light environments at the water surface. It is alsoreported that microbial fuel cells in current designs can be made morerobust if powered by solar energy, and the power possible in suchsystems depends on the nature of the nitrogen source and theavailability of light. In carefully designed systems a combination ofnitrogen (e.g. from sediments, wastewater, or agricultural wastes)processing systems can be powered using solar energy to power thenitrogen processing cells of the system (see Cho et al, J. AppliedMicrobiology 104 (2008) 640) with the result yielding more electricityand increased longevity of the system.

Thus, devices using the oligonucleotide attachment systems of theinvention can be made such as, for example, fuel cell devices using theoligonucleotide modified non-animal cells of the invention and otherprinciples consistent with the invention. For example, hydrogenproducing fuel cells can be made using modified plant cells. Typicallythe plant cells can be patterned so that hydrogen producers and oxygenscavengers are combined in an optimal arrangement for energy production.The devices can be planar or spherical, or provide any surface optimalfor the capture and processing of solar energy and can comprise asensor. Devices can also be made to study or use for energy purposesplant, bacterial or algae cells. The device can further be used to storeenergy. The device can comprise a sensor, or can be a bioreactor. Algaebased systems can be a foundation for sustainable and commerciallyviable integrated biological hydrogen production processes usingphotosynthetic H₂ production by green algae, the visible region of thesolar spectrum coupled to H₂ production by anoxygenic photosyntheticbacteria utilizing the near infrared region of sunlight. Biomassaccumulation in the course of photosynthesis by the two organisms issubsequently utilized in dark anaerobic fermentations for further H₂production. Small organic acids accumulate as a by product of the darkanaerobic fermentation, and these can serve as a substrate to supportfurther hydrogen production by green algae and photosynthetic bacteria.The foundation of such integrated H₂ production is the oxygenicphotosynthesis of unicellular green algae (e.g. Chlamydomonasreinhardtii), a process that uses the energy of sunlight to convertwater, carbon dioxide and other inorganic nutrients into the basicbuilding blocks of life. (See A. Melis, and M. R. Melnicki,International J. of Hydrogen Energy 31 (2006) 1563-1573).

Other photosynthetic, photovoltaic non-animal cell based systems towhich the tools of this invention can be applied include Dickson et al.International J. Hydrogen Energy, 34 (2009) 204-215 which describes useof Synechocystis sp. PCC 6803 to form a silica sol-gel. Such technologymay be directly applicable to powering small electronic devices. Sui etal. J. of Microelectromechanical systems vol. 17, no. 65, December 2008describes a microfabricated polydimethyl-siloxane (PDMS) microbial fuelcell (MFC) with embedded micropillar electrodes. This MFC ischaracterized by a flexible and biocompatible structure suitable forbody implantation as a potential power source for implanted bioMEMSdevices. Song et al, describes a microfluidic polymer electrolytemembrane (PEM) fuel cell using polydimethylsiloxane (PDMS) for a bioMEMSdevice, which can be used for powering portable electronic devices andother miniaturized MEMS devices. The work described by Song et al,patterns a submicron-thick Nafion membrane on a glass substrate using areversibly bonded PDMS microchannel to generate an ionselective membranebetween the fuel cell electrodes, instead of sandwiching a thin Nafionsheet. Due to the flexibility of the PDMS material, the Nafion membranecan be sealed between the PDMS chip and glass substrate by oxygen plasmabonding. Surface patterning can be seamlessly integrated into a standardmicrofabrication process flow and form a PEM microfluidic fuel cellwithout cumbersome clamping of the layers together and inherent risks ofleakage due to ineffective clamping. At the very least, this deviceconstruction makes possible building of massively parallel arrays ofmicrofluidic fuel cells. Patterning of cells can be facilitated usingthe cell attachment methods of the present invention.

In another embodiment, the cell surfaces that are modified using thepresent modification technique are cell membrane-like surfaces such asthe coat proteins of viruses, phage and other self-assembledbiomolecular surfaces or structures. Example 9 demonstrates that viralor phage capsids modified with the present cell modification methods toconjugate a DNA aptamer to the surface, enables the creation ofmultivalent cell targeting vehicles. Thus the present direct cellmodification methods can be used in the modification of many monomericor biomolecular surfaces for any number of applications requiring thedirect attachment of a nucleic acid or oligonucleotide.

V. Kits

The present invention also provides kits having an activated nucleicacid moiety suitable for forming a nucleic acid-cell conjugate of thepresent invention, and a substrate surface having a complementarynucleic acid moiety. In some embodiments, the present invention providesan activated nucleic acid moiety suitable for covalent linkage to anative functional group of a cell surface, and a substrate surfaceincluding a nucleic acid moiety complementary to the activated nucleicacid moiety. The activated nucleic acid moiety can be a powder or be ina solution. The kit can also include buffer solutions known to one ofskill in the art, and other solutions for forming the nucleic acid-cellconjugate and then binding the nucleic acid-cell conjugate to thesubstrate surface via the nucleic acid moiety on the substrate surface.

The components of the kits are described in more detail above. Thesubstrate surface can include any suitable material including, but notlimited to, glass microscope slides, cover slips, tissue culture plates,or well plates. The substrate surface can be preprinted with nucleicacid moieties in specific locations. The kits can include othercomponents, such as fixing solutions for the cells, solutions ofdetection agents, solutions of cell staining agents, buffer solutions,etc. Other solutions can include the nucleic acid moieties themselves,and solutions and reactants for attaching the nucleic acid moieties tothe cells.

VI. Examples Example 1 Conjugation of DNA to a Cell without a Cell WallVia a Lysine Native Functional Group

All cell culture reagents were obtained from Gibco/Invitrogen Corp(Carlsbad, Calif.) unless otherwise noted. Cell culture was conductedusing standard techniques. Jurkat cells were grown in T-25 cultureflasks (Corning, USA) in RPMI Medium 1640 supplemented with 10% (v/v)fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycin (P/S,Sigma). MCF-7 cells were grown in DMEM supplemented with 1%non-essential amino acids and 10% fetal bovine serum, plus 1%penicillin/streptomycin. MDA-MB-231 cells were grown under the sameconditions as the MCF-7 cells, but without non-essential amino acids.

Fluorescence micrographs were acquired using an Axiovert 200M invertedmicroscope (ZEISS) with fluorescence filter sets for DAPI/Hoechst,fluoroscein/fluo-3, and rhodamine. Ultraviolet absorption of thedifferent oligonucleotides was determined at 260 nm on a UVIKON 933double beam UV/vis spectrophotometer (Kontron Instruments, UnitedKingdom).

Synthesis of NHS-DNA Conjugates.

For cell adhesion studies, three complementary oligonucleotide pairswere designed such that they were identical in overall composition anddiffered only in sequence. Each sequence pair was also calculated topossess comparable melting temperatures (55° C.) and minimal secondarystructures.

The sequence identities were as follows:

The sequence identities were as follows:

C1: (SEQ ID NO: 1) 5′-GTA ACG ATC CAG CTG TCA CT-3′ M1: (SEQ ID NO: 2)5′-AGT GAC AGC TGG ATC GTT AC-3′ C2: (SEQ ID NO: 3)5′-TCA TAC GAC TCA CTC TAG GG-3′ M2: (SEQ ID NO: 4)5′-CCC TAG AGT GAG TCG TAT GA-3′ C3: (SEQ ID NO: 5)5′-ACT GAC TGA CTG ACT GAC TG-3′ M3: (SEQ ID NO: 6)5′-CAG TCA GTC AGT CAG TCA GT-3′

The oligonucleotides were obtained from Integrated DNA Technologies(Coralville, Iowa) with thiol groups installed at the 5′-end. Samples (2mg in 80 μL) were combined with 320 μL of 10 mMtris(2-carboxyethyl)phosphine (TCEP) and 400 μL of 1×TE buffer (10 mMTris with 1 mM EDTA, brought to pH 7.5 with HCl) and stored frozen at−20° C. until use. NHS-PEO₆-Maleimide(succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol] ester) waspurchased from Pierce. A stock solution was prepared by dissolving 5 mgof NHS-PEO₆-maleimide in 1 mL of DMSO (Sigma). Aliquots of this solution(20 μL each) were stored at −20° C. until use.

DNA modification was achieved by passing a thawed solution of 5′-thiolssDNA (30 μL, 0.39 mM) through a NAP-5 size-exclusion column (GEHealthcare). The eluent was then exposed to 20 μL of theNHS-PEO₆-Maleimide solution at room temperature for 10 minutes. Thereaction was then purified by passing it through a second NAP-5 columnthat was pre-equilibrated with PBS solution (pH 7.2). The concentrationof DNA in the column eluent was verified using UV-vis spectroscopy. Theresulting solution was then applied to samples of live cells.

To confirm the nature of the modification chemistry, models of theoligonucleotide conjugates were prepared and characterized. To do this,0.5 mL of DMF was saturated with 6-amino-N-(4-aminophenethyl) hexanamideand added to 1 mL of the reaction solution obtained after NAP-5purification. After 30 min of incubation at room temperature, theoligonucleotide conjugates were analyzed using MALDI-TOF MS. Observedmasses were within 0.090% of expected values.

Modification of live cells and quantification of attached DNA moleculeswas accomplished as follows: Immediately prior to modification, a sampleof 5×106 Jurkat cells was washed with PBS buffer three times to removeany proteins from the culture medium. The cells were then exposed tosolutions of NHS-DNA (C3 strand, 3 μM to 54 μM final concentrations) for30 minutes at room temperature. After isolation via centrifugation, thecells were returned to the culture medium, or labeled with fluorescentDNA complements as described below.

In order to quantify the number of surface DNA molecules, portions ofthe modified cells were incubated with 10 μL solutions of FITC-labeledcomplementary DNA strands at 0° C. for 30 minutes. The cells were thenwashed with PBS solution and resuspended in PBS containing 1% FBS priorto analysis. The cells were then analyzed by flow cytometry.Fluorescence measurements were calibrated using fluorescent beads ofknown fluorophore density. Fluorescence measurements were compared tothose corresponding to control cells lacking DNA modification and cellsreacted with mismatch DNA sequences.

FIGS. 31A and 31B show the DNA-cell conjugates prepared by the methodabove binding to substrate surface modified with a complementary DNAstrands, demonstrating formation of the DNA-cell conjugate.

Example 2 Conjugation of DNA to a Cell without a Cell Wall Via anAspartic Acid or Glutamic Acid Native Functional Group

DNA has been conjugated to cells using EDC coupling chemistry by thefollowing procedure:

1. Take 5 million cells and wash them with 10 mL PBS two times.

2. Dissolve 0.366 g EDC in 19.06 mL PBS.

3. Add 0.452 g NHS to the EDC solution.4. Make sure pH is around 7. (Normally they are)5. Take 1 mL of this EDC/NHS solution, added with 200 uL 80 uMamine-ssDNA (in 1×SSC).

6. Incubate at rt for 30 min to 1 hr.

7. Wash with 10 mL of 1% FBS/PBS three times.8. Resuspend to 50 to 100 uL 1% FBS/PBS, and ready to be applied to theslides.

Example 3 Conjugation of DNA to Oxidized Sugar Native Functional Group

Oxidation of Surface Exposed Diols (Lipopolysaccharide, LipoteichoicAcid, Etc.) on Cells with Cell Walls.

Rinse cells (10⁵-10⁷) in 1 mL Dulbecco's phosphate buffered saline(DPBS) using centrifugation. Suspend cells in 2 mL 0.5-5 mM NaIO₄ inDPBS and place at 37° C. with orbital shaking for 20 min. Lowerconcentrations of periodate are needed for eukaryotes such as algae andyeast. Neutralize periodate with 1 mL 0.1 M glucose in DPBS. Centrifugecells down and wash 2×1 mL DPBS, 1×1 mL pH 6 MOPS buffer (see below).

Attachment of Hydrazide-Modified ssDNA to Oxidized Surface ExposedDiols.

Suspend cells in 800 μL pH 6 MOPS Buffer (0.5 M NaCl, 0.1 M3-(N-morpholino)propanesulfonic acid, 18.2 MΩ H₂O, adjust pH to 6.0)+10mM aniline+˜35 μM hydrazide ssDNA (purchased from Integrated DNATechnologies). Allow cells to react at room temperature for 1-18 hr,depending on concentration of surface diols, with gentle shaking. Spincells down and rinse with DPBS

FIGS. 32A and 32B show the DNA-cell conjugates prepared by the methodabove binding to substrate surface modified with a complementary DNAstrands, demonstrating formation of the DNA-cell conjugate.

Example 4 Devices

General protocol for the attachment of DNA strands to cells and fortheir immobilization onto DNA-printed surfaces was as follows:Immediately prior to modification, a sample of 5×106 Jurkat cells waswashed with PBS buffer three times to remove any proteins from theculture medium. After the final rinse, additional PBS was added to bringthe volume to 5 mL (1×106 cell/mL). The cell suspension was then reactedwith 1 mL of NHS-DNA (11.7 μM) solution synthesized and purified from 30μL of 5′-thiol ssDNA (C2 sequence). The mixture was allowed to react atroom temperature for 30 minutes, and was then washed three times withPBS containing 1% FBS. The cells were then resuspended in 0.5 mL of PBScontaining 1% FBS.

To print the glass surfaces, a 20 μM solution of 5′ amine functionalizedssDNA in 3× saline sodium citrate buffer (SSC: 45 mM sodium citrate, 450mM NaCl, pH 7.0) was used for sample preparation. DNA solutions weredeposited onto aldehyde-functionalized glass slides (SCHOTT Nexterion,Louisville, Ky.) by manual pipetting or by using a robotic microarrayprinting system at the UC Berkeley Functional Genomic Laboratory.Spotted DNA was immobilized and the slides were passivated according tothe manufacturer's protocol. After printing, the slides were dried undera stream of N₂ and stored in the dark under a dry atmosphere. Patternedslides were typically used within one month.

Micropatterning of the glass slides was achieved using photolithographyin conjunction with an aluminum lift-off technique, and will bedescribed in full detail elsewhere. For studies with one cell type, allcells were labeled with the C2 sequence. Slides were patterned withcomplementary DNA sequence, M2 unless otherwise noted. Solutions ofDNA-modified cells were introduced onto each surface and incubated for3-5 minutes without agitation. The devices were then washed twice withPBS containing 1% FBS. Replicate data sets were collected by selectingthree device regions at random before washing. Each location wasphotographed, washed, and then visualized again.

Evaluation of cell viability was made as follows: Jurkat cells coatedwith the C2 strand were seeded in a 1 mL Petri dish with normal growthmedia, and M2 strand DNA was added into the solution to a concentrationof 2 μM. Unmodified Jurkat cells were cultured under identicalconditions as a control. Cells were counted in each of the four samplesusing a hemocytometer at 24, 48, and 72 hours. Cell viability wasmonitored by adding Trypan Blue.

The extent of apoptosis of surface bound cells was determined by annexinV/propidium iodide staining (BD Biosciences). After immobilization onthe slides by DNA, the cells were incubated in normal media at 37° C.for 24 hours. A sample of unbound Jurkat cells (lacking surface DNAstrands) was incubated under the same conditions as a control. Asolution consisting of 900 μL of 1× binding buffer, 30 μL of the annexinV-FITC stock solution, and 30 μL of the PI stock solution was prepared.After 24 hours, 100 μL of this solution was applied to the slides for 15min at room temperature. The cells were imaged by fluorescencemicroscopy and the number of non-viable cells counted after one hour.

Immobilization of adherent cell lines on patterned surfaces wasaccomplished as follows: Two breast cancer cell lines, MCF-7 andMDA-MB-231, were obtained from ATCC. The cells were detached fromculture plates with 1 mM EDTA without any trypsin, and the cellsolutions were washed with PBS three times. A 5 mL portion of the cellsolution (1×10⁶ cell/mL) was reacted with 1 mL of NHS-DNA solutionsynthesized from 30 μL of 5′-thiol ssDNA (C2 sequence) as describedabove. The mixture was allowed to react at room temperature for 30minutes, and was then washed three times with PBS containing 1% FBS. Thecells were then resuspended in 0.5 mL of PBS containing 1% FBS. The cellsolution was introduced onto glass slides patterned with complementaryDNA sequence M2, and the samples were incubated for 5 minutes. Theslides were then washed two times with PBS containing 1% FBS. Afterimmobilization onto slides via DNA hybridization, the cells wereincubated in their normal media and observed for 36 hours. Replicatedata sets were collected by photographing three different surfaceregions at 12 hour intervals.

Confirmation of the sequence-specificity of cell immobilization was madeas follows: DNA-modified Jurkat cells and MDA cells were prepared byincubating each cell population with NHS-DNA (sequence C2 or C1,respectively) in PBS for 30 minutes as described above. To facilitatevisual differentiation of the cells, the cytosol of each population waslabeled with either CellTracker Blue™ or CellTracker Green™ live cellstains. After rinsing, equal amounts of each population were mixed,introduced onto microspotted DNA microarrays bearing either sequence M2or M1 (constructed as above), and incubated for 5 minutes. Themicroarray was then washed twice with PBS containing 1% FBS and observedunder a fluorescence microscope.

Immobilization of human red blood cells was accomplished as follows:Fresh samples of red blood cells were obtained from a blood sample of ahealthy human and stored in 1% citric acid solution at room temperature.Cells were used within 1 hour. The cell solution was washed three timeswith PBS and was then incubated in the NHS-ssDNA solution for 30 minutesto allow modification of cell surfaces. The cell suspension was thenwashed three times with 1% FBS/PBS solution before being applied toglass slides bearing the complementary ssDNA strands. After cellattachment, the glass slides were washed with 1% FBS/PBS to remove anyunbound cells and viewed under an optical microscope. Cells wereincubated in 1% FBS/PBS after immobilization, and their viability wasexamined after 3 hours using trypan blue staining.

Patterning of primary CD4+ T cells and IL-2 Production Assay wasconducted as follows: Primary CD4+ T cells (obtained in collaborationwith Jay T. Groves' lab, UC Berkeley) were harvested from mice and grownunder reported conditions before use. The primary T cells were thenmodified using the NHS-DNA protocol and exposed to different DNApatterns printed by spotting or by using photolithography, as describedabove. The glass slides with DNA-immobilized cells were washed with 1%FBS/PBS to remove any unbound cells and viewed under a microscope.

The IL-2 production of primary T cells immobilized with DNA duplexes wasexamined using ELISA. A population of 2×10⁵ primary T cells was modifiedwith DNA strands and immobilized on a series of slides (1 cm²) bearingthe complementary sequence. These samples were then divided into threeportions. The first sample was incubated in normal T cell growth mediawithout any additional reagents. The second sample was treated with PHA(1 μg/mL) and PMA (50 ng/mL). The third sample was treated with ConA (1μg/mL), PMA (50 ng/mL) and CSA (μg/mL). Analogous samples of free Tcells with no surface DNA were prepared as controls. All the cellsamples were incubated at 37° C. for 20 h and then centrifuged. Portionsof the culture media (1 mL) were withdrawn from each population of cellsand tested for IL-2 production using a Mouse Interleukin-2 ELISA testkit (Thermo Scientific).

Patterning of primary myoblasts was accomplished as follows: Primarymyoblasts (obtained in collaboration with Randall Lee's lab, UCSF) wereharvested from mice and purified according to a published protocol asdescribed in Huang, N. F.; Patel, S.; Thakar, R. G.; Wu, J.; Hsiao, B.S.; Chu, B.; Lee, R. J.; Li, S. Nano Lett. 2006, 6, 537-542, herebyincorporated by reference. Briefly, normal cell growth was achieved inHam's F-10 media (Invitrogen) with 10% (v/v) fetal bovine serum (FBS,HyClone), 1 bGF (Invitrogen), and 1% penicillin/streptomycin (P/S,Sigma). Immediately before surface modification the cells were detachedwith 1 mM EDTA without any trypsin. The resulting cell suspensions wererinsed with PBS three times. A 5 mL portion of the cell solution (1×10⁶cell/mL) was reacted with 1 mL of NHS-DNA solution. The mixture wasallowed to react at room temperature for 30 minutes, and the cells werethen washed three times with PBS containing 1% FBS. Surfaces werepatterned with the complementary DNA sequence through spotting orphotolithography, and incubated with PBS containing 1% FBS at roomtemperature for 1 h. The cell solution was introduced onto the slidesand incubated for 5 minutes. The devices were then washed three timeswith PBS containing 1% FBS. After immobilization, the cells wereincubated in growth media or fusion media (DMEM (Invitrogen) containing5% horse serum, and 1% penicillin/streptomycin (P/S, Sigma)) at 37° C.for 14 days. The unbound myoblasts were cultured under identicalconditions as a control. Images and movies of all cell samples wererecorded every 24 hours.

Example 5 DNA Barcode

A microdevice is developed for DNA-barcode directed capture of singlecells on an array of pH-sensitive microelectrodes for metabolicanalysis. Cells are modified with membrane-bound single-stranded DNA,and specific single-cell capture is directed by the complementary strandbound in the sensor area of the iridium oxide pH microelectrodes withina microfluidic channel. This bifunctional microelectrode array isdemonstrated for the pH monitoring and differentiation of primary Tcells and Jurkat T lymphoma cells. Single Jurkat cells exhibited anextracellular acidification rate of 11 milli-pH/min, while primary Tcells exhibited only 2 milli-pH/min. This system can be used to capturenon-adherent cells specifically and to discriminate between visuallysimilar healthy and cancerous cells in a heterogeneous ensemble based ontheir altered metabolic properties. The bifunctional microelectrodearray demonstrated here shows how devices made using the composition ofthis invention have the ability to selectively capture cells and measuretheir electrical and metabolic activity. Using DNA-barcode capture, bothadherent and naturally non-adherent cells can be studied on the samedevice. In addition, the cells so captured are not activated. The arrayformat allows direct discrimination between cells from a mixture,revealing the variation in single cell properties and how that cellcontributes to the whole. Controlled single-cell electrochemicalmeasurement points to using a nanoscale cell interface to enablemultiplex subcellular analysis of cellular activity.

The controlled capture of single cells in microfluidic devices isessential for the development of integrated microdevices for single cellanalysis. With size and volume scales comparable to those of individualcells, microfluidic devices provide a powerful tool for control of thecellular microenvironment. It has been demonstrated that the use ofengineered cell surface DNA (cell adhesion barcodes) is capable of cellcapture, prior DNA capture of cells could not be used to performsingle-cell gene expression analysis in a microfluidic chip, and havethat gene analysis representative of an unactivated cell. The devices ofthis invention have the new capacity to employ DNA barcode cell captureto populate an array of pH-sensitive microelectrodes with unactivated(native) cells. The system enables rapid, selective and reversiblecapture of non-adherent single cells (previously impossible), as well asadherent cells, on the pH sensor surface. This bifunctional systemenables accurate real-time monitoring of single cell metabolism based inthe principle that extracellular acidification is proportional tooverall energy usage. In these experiments it is demonstrated that thistechnology can identify cancer cells with high metabolic activity,making the device a potential diagnostic and prognostic tool for cancertreatment. Additionally, because the system can be tailored to beextremely selective and specific about the cells studied and these cellscan be placed at precise locations on the device surface, the system isideal for applications known as patient specific analysis and treatmentfor any disease, including of course cancer.

Previous work has demonstrated the individual aspects of single cellcapture and pH monitoring in microfluidic systems. A variety of methodsfor arrayed single cell capture have been shown, including physical andenergetic traps, and biochemical adhesion. The cells in this prior workwere not captured using DNA, but rather physically restrained bybarriers in the device environment. While a simple restrictive capturewell or microfluidic trap could be used to isolate cells over a sensor,it has been shown that access to fresh media and the ability to clearwaste products are important to normal cell function. Highly precisecell placement is also important for monitoring activity ifsubcellular-scale electrodes are to be used. The oligonucleotideattachment system of the present invention allows for both captureirrespective of physical barriers on the device, and feeding and washingthe cells without risk of losing them in the process.

The use of extracellular acidification is a valuable tool in thequantitative analysis of cell activity. A key example is the CytosensorMicrophysiometer, which has been widely used to measure acidificationfrom bulk cell populations (10⁴-10⁶ cells per 3 ul sample) as a way toquantify metabolism. This system has been used for a number ofapplications, including the detection of G-protein coupled (chemokine)receptor activation, neurotrophin activity, ligand gated ion channels,and the binding of ligands to tyrosine kinase receptors. It has alsobeen used to identify ligands for orphan receptors. Other devices havealso employed pH electrodes to measure cell activity down to the singlecell level. Work described in Ges et al. Biomed. Microdevices, 2008, 10,347-354 recently demonstrated a device for on-chip measurement ofacidification rates from single cardiac myocytes using physicalconfinement. In the Ges system, single myocytes were isolated in thesensing volume by physically pinching closed the ends of a PDMS channel.While this system represents an important step in single cellmonitoring, the cell isolation technique does not allow for controlledcapture on the sensor electrodes, which is necessary for simultaneousmulti-analyte monitoring from single cells in the same controlledenvironment. The present invention provides these significantadvantages.

The devices described here provide direct integration of theoligonucleotide-based cell capture technique with sensors that are onthe same size scale of the individual cells, thus allowing cell analysisnever before achievable in a bifunctional electrode system. An array oflithographically patterned iridium oxide pH microelectrodes is enclosedwithin a microfluidic channel. Single stranded DNA is attached to theiridium oxide surface using a silane linker, giving the sensor theability to capture cells bearing complementary DNA while retaining itsdetection sensitivity. Here we use this system to measure theextracellular acidification resulting from the metabolism ofnon-adherent T cells, and we demonstrate that the pH sensitivity issufficient to discriminate between healthy primary T cells and cancerousJurkat T cells that have a higher metabolism. Our results demonstratethe differentiable metabolic activity of individual healthy andtransformed cells of the same basic type, which could enable theidentification of circulating tumor cells (CTCs) within a heterogeneoussample. The novel combination of DNA-directed cell capture andelectrochemical monitoring on a bifunctional electrode offers a newplatform for single cell analysis.

Electrode sensor fabrication is accomplished as follows: Electrodes (40nm thick Au with a 20 nm Cr adhesion layer) were patterned on 1.1 mmthick borofloat glass wafers using standard photolithographic liftoff,as previously described (FIG. 11). A 7 μm thick layer of Parylene-c wasdeposited on the wafer using a Specialty Coating Systems Labcoter 2Parylene deposition system, and measured with an AlphaStep IQprofilometer. A 100 nm layer of aluminum was evaporated on the device,and then lithographically etched using Air Products aluminum etchantwith surfactant for 30 s at 60° C. (FIG. 11A, 11B). The etch mask forthe aluminum layer was a photolithographically patterned 1 μm thick filmof Shipley 1818 photoresist. The aluminum layer was then used as a maskto etch the underlying Parylene using oxygen plasma (60 sccm O₂, 100 W,60 min).

After removing the Parylene insulation from the sensor area, the sensorswere electro-deposited with a layer of iridium oxide following theprotocol of Yamanaka et al. Briefly, the iridium deposition solution wasprepared as follows. 37.5 g of IrCl₄ was added to 75 mL of de-ionizedwater and stirred for 90 min. Next, 125 mg of oxalic acid was added, andthe solution was stirred for 3 h. Finally, the solution pH was adjustedto 11 using K₂CO₃. The solution was initially light yellow, turninglight blue, and finally dark blue over the course of several weeks. Thedeposition solution was stable for at least six months afterpreparation. Iridium oxide deposition was performed using a CHI 660potentiostat in voltage cycling mode. 240 cycles of +0.7 V (0.25 s) and−0.5 V (0.25 s) were used, in a three electrode configuration using asaturated calomel reference and a platinum counter electrode.

After deposition, the devices were plasma cleaned for 1 min and modifiedwith trimethoxysilylpropanal by vapor deposition at 60° C. for 60 min.Amine-modified ssDNA (80 μM in phosphate buffered saline) was thendeposited onto the devices and bound using reductive amination aspreviously described (FIG. 11C). Following DNA deposition, theprotective aluminum layer was dissolved by treatment with 0.1 M NaOH atroom temperature with stirring for 20 min, leaving the capture DNA onlyon the sensor surface (FIG. 11D).

Microfluidic device preparation is accomplished as follows:Poly(dimethylsiloxane) (PDMS) channels were prepared using Dow CorningSylgard 184 with SU-8 or polystyrene molds. Channels were 5 mm wide, 15mm long, and 600 μm in height. A fluidic inlet compatible with 20 gageTeflon tubing was punched using an 18 gage blunt-tipped needle, and a 5mm diameter outlet reservoir was punched on the other end. PDMS channelswere cleaned with a UV/ozone system for 10 min, and applied to thedevice. The channels were filled with DI water for 1 h to allowhydration of the iridium oxide layer, then the pH response of theelectrodes was calibrated using standard pH 4, 5, 7 and 10 buffers. Thechannel was maintained at 37° C. using a heated aluminum stage with aMinCO polyimide heater and Cole-Parmer DigiSense PID temperaturecontroller.

Cell preparation and labeling is accomplished as follows: Jurkat cellswere cultured in RPMI-1640 media with 10% fetal bovine serum (FBS) and1% penicillin-streptomycin solution. Cultured cells were maintained at37° C. in 5% CO₂, and split 1:10 every 2-3 days. Cell acidificationexperiments were conducted in custom low-buffered media bases onDulbecco's Modified Eagle's Medium, containing 25 mM D-glucose, 5.3 mMKCl, and 110.34 mM NaCl, plus 1% FBS and penicillin/streptomycin.Finally, the media was pH adjusted to 7.45 using 0.1 M NaOH. Cells wereisolated from mice and prepared as previously described (see A. L.DeMond, K. D. Mossman, T. Starr, M. L. Dustin and J. T. Groves, Biophys.J., 2008, 94, 3286-3292).

Cell-surface labeling with ssDNA was achieved using an NHS-DNA conjugatethat covalently modifies primary amines on the cell surface (FIG. 11E),as described above. Briefly, cells were incubated in a 120 μM NHS-DNAsolution in PBS at room temperature for 30 min, then washed three timesto remove any unbound DNA. Barcode-specific cell capture was tested withspotted DNA microarray slides as previously reported (see Douglas et al.Lab Chip, 2007, 7, 1442-1448).

Metabolic monitoring was accomplished as follows: Cells were suspendedat a concentration of 10⁶/ml, and the suspension was flowed into themicrofluidic device. Cell suspensions were flowed into the channel using1 mL syringes with Teflon tubing. Where Jurkat and primary T cells weremonitored simultaneously they were labeled with CellTracker Green andRed dyes, respectively, as previously described and mixed in an equalratio. Following a 5 min incubation to allow DNA-based cell capture, theunbound cells were rinsed away (5 ul/min for 3 min) with thelow-buffered media. After rinsing, the pH response was monitoredelectrochemically for 10 min. After this recording, cells were releasedfrom the electrodes by heating the device to 55° C. and applying astrong rinse (200 uL/min) with the low-buffered media. Once rinsed andallowed to return to 37° C., the device could be reloaded with cells.This allowed for multiple measurements to be taken with a single cellpreparation.

Voltage measurements were recorded between the iridium oxide electrodeand a distant FLEXREF Ag/AgCl reference electrode from World PrecisionInstruments. An identical iridium oxide electrode outside the cell areawas used to compensate for any sensor drift. The sensor electrodes wereconnected to a National Instruments PCI-6031E data acquisition card with16 bit analog to digital conversion. The digitized signals weremonitored using a custom Labview VI, sampling in multiplex at 3 hz.Voltage signals were processed with a 1% Loess filter using Peak Fitsoftware to reduce noise.

Before metabolic analysis, the electrodes were characterized usingstandard pH buffers (FIG. 12). These DNA-modified electrodes were foundto retain their pH sensitivity, with performance comparable tounmodified iridium oxide sensors. The electrode response was stable andfast, responding to a 1 pH unit change in under 500 ms. The pH responseof the electrodes was typically −68.5 mV per pH unit, with a linearresponse over the range pH 4 to 10. The typical range for cellacidification measurements is approximately 6.5 to 7.5, so this sensoris well suited for the measurements. The magnitude of the observedresponse is in line with the −60 to −80 mV/pH range of other hydratediridium oxide sensors previously demonstrated. The reaction at theelectrode that provides the pH sensitivity has been described by Olthuiset al. The −60 to −80 mV/pH sensitivity range is dependent on theoxidation state of the iridium oxide film as deposited by variouselectrochemical techniques. The reaction at the electrode that providesthe sensitivity was provided by Olthuis.

2Ir(OH)₂O⁻+H₂O═Ir₂O(OH)₃O³⁻+3H⁺+2e ⁻

The integration of an affinity capture DNA probe with the pHmicroelectrodes on the bifunctional microelectrode array chip provides aplatform for the direct monitoring of extracellular acidification forcells that are normally non-adherent. The system also providesmonitoring of cells that are not activated by the oligonucleotideattachment system, whether adherent or non-adherent cells. As seen inFIG. 13, the size-limiting bifunctional microelectrode enables singlecell capture directly on the sensor. The bifunctional microelectrodearray was tested by measuring the extracellular acidification of Jurkatand primary T cells. First, Jurkat and primary T cells were captured andmonitored separately on the array to establish the sensor functionalityand the difference in single-cell acidification between the two celltypes. FIG. 14A shows single cell acidification data over a 10 minperiod. Jurkat cells exhibited an extracellular acidification rate of11.5±3.3 milli-pH/min, while primary T cells exhibited 1.61±1.5milli-pH/min (s.d., n=9 each). This difference was also confirmed withbulk cell population acidification measurements (˜10⁶/ml cells inlow-buffered media at 37° C.).

To demonstrate the ability to distinguish different cells in a mixedpopulation, single cells from a mixture of Jurkat and primary T cellsbearing the same cell adhesion barcode were monitored simultaneously onthe array. FIG. 14B shows acidification data from mixed cells on thearray over 10 min. The difference in measured acidification ratesfollowed the same trend as the separate samples, and allowed fordiscrimination between the two visually similar cells (FIG. 14B). Jurkatcells had an acidification rate of 10.1±2.3 milli-pH/min, and healthy Tcells had 2.41±2.54 milli-pH/min (s.d., n=5 each).

FIG. 14C presents a bar graph of the acidification rates over severaltrials using known cell populations on the array. For Jurkat cells themean acidification rate was 11.5±3.2 milii-pH/min, while primary T cellsexhibited a rate of 1.62±1.31 milli-pH/min. The difference is clearlysignificant with a T-test value of P<0.0002. While the Jurkat cells wereslightly larger than the primary T cells (typically 12 μm vs. 10 μmdiameter), the size difference is not large enough to account for thedifference in acidification.

To demonstrate the ability to measure single cell response to exogenousstimulation, Jurkat cells were treated with rotenone while captured onthe bifunctional microelectrode array (FIG. 15). Incubation withrotenone would be expected to interfere with the mitochondrial electrontransport chain, causing cells to shift to lactic acid fermentation tocomplete the glycolytic cycle. The resulting excretion of lactic acidshould then increase the rate of acidification in the cellularenvironment. In the experiment, captured cells were first incubatedunder normal conditions to establish a baseline rate of acidification(˜8.8 milli-pH/min) under aerobic metabolism. After 13 min 10 μMrotenone was added to the channel, which resulted in a three-foldincrease in the acidification rate (˜27.7 milli-pH/min) within 1 minute.Bulk cell controls, in which Jurkat cells were treated with 1 μMrotenone in low-buffered media (˜10⁶ cells/mL at 37° C.), consistentlydemonstrated more than twice the acidification over 60 min compared toidentical untreated cells. The observation of this metabolic shiftprovides an important demonstration of this technique's ability tomonitor responses to exogenous agents, such as receptor-ligand binding,at the single cell level.

The bifunctional microelectrode array developed here combines the twoimportant functions of selective cell capture and metabolic monitoringof single cells in an array format. In earlier work, Castellarnau et al.used dielectrophoresis to localize high concentration suspensions ofbacteria near an ISFET pH sensor and measured the acidification of thecells in the presence of glucose. While this technique was well suitedto measurement of the bulk response, it lacks the ability to resolve theunique activity of single cells. The single cardiac cell pH system ofGes et al. provides the ability to monitor large adherent cells, but thevolume displacement caused by sealing the channel makes it difficult todirect the cell attachment. DNA-barcode capture provides the advantageof directed capture of both adherent and naturally non-adherent cells,such as T and B cells, and the additional advantage of analyzing cellsthat are not activated by the oligonucleotide attachment method. Thiscontrolled capture provides a platform for spatially-resolved electricaland/or optical probing and measurement of activity on the cell surface.

The acidification data show that single non-adherent cells continue tobehave normally after treatment with capture DNA and attachment to theelectrode. While any capture technique is likely to have some effect onthe cell, cell adhesion barcodes bypass the natural cell-surfacereceptors that are often used for integrin or antibody-based capture,and should thus avoid the activation of those known signaling pathways.For both the Jurkat and primary T cells the extracellular acidificationrates measured are comparable to the single cell acidification ratesreported by Ges et al., but the increased sensitivity of ourfunctionalized microelectrode technique allows discrimination betweenthe two cell types.

Our single-cell results show that the difference between the metabolicactivity of primary non-transformed cells and immortalized cancerous Tcells can be detected at the single-cell level. We have demonstrated theability to electrochemically distinguish between visually similar singlecells from the same basic type using this metabolic difference. Thismethodology could be used to identify individual circulating tumor cellsby their distinctive metabolic activity, going beyond simpleantibody-based capture. It could also be used to differentiate betweencancerous cells of different metastatic potential. Single-cellmonitoring within such a mixture would allow for the detection ofdifferences in drug response based on the cell's state of cancerprogression or origin.

The array format with its obvious extension to include more elementsallows the direct comparison of the individual activity of many cellsunder the same conditions with sufficient power to characterize ensemblevariation. The construction of a nanofabricated electrode array couldproduce an electrochemical analysis map of a cell surface with highspatial-resolution. Static cell surface profiling has previously beendemonstrated using scanning electrochemical microscopy, but ananoelectrode array could transform this from a serial to a parallelprocess and provide temporal resolution as well in live cells.

The system is capable of increasing the number of detected analytes froma single cell and has the capacity for increased complexity of theanalysis system in general. The previously mentioned CytosensorMicrophysiometer system for bulk cell monitoring was modified tosimultaneously measure glucose, lactate and oxygen levels, in additionto the standard pH measurement capabilities. Micro- or nanofabricatedanalyte-selective sensors could also be added to the system foradditional analytical depth, including multi-analyte sensing on a singlecell. A combination of calcium-sensitive fluorophores and electricalcontrol has been used to monitor calcium flux in single neurons duringpatch-clamp recording by Thayer et al. The PDMS/glass multilayer deviceis readily modified to enable simultaneous fluorescence and electricalmeasurements. While fluorescent probes often suffer from photobleaching,the technique embodied here could be used to track single-cell metabolicactivity over hours or days, revealing any changes as the cellprogresses through its life cycle.

DNA barcode-based cell capture provides the ability to engineerattachment between individual cells which allows for the constructionand analysis of discrete multi-type cell systems on an electrode. Forexample, a single neuron could be linked using DNA to a single musclecell to allow analysis of the single-cell neuromuscular synapticformation and operation. Additionally, artificial tissue can beengineered using cell to cell attachment with oligonucleotidehybridization. Sensors can be placed in or near the system to recordmetabolic, electrical or other changes in the system.

Example 6 Microfluidic Bioprocessor

Single-cell analysis is a powerful approach for understanding changes ingene expression within an isogenic cell population. Traditional geneexpression analysis techniques such as microarrays and serial analysisof gene expression are not sensitive enough to analyze changes at thesingle-cell level and only report on the ensemble average behavior of alarge numbers of cells. Recently, a variety of highly sensitive andspecialized techniques have been developed for probing gene expressionin single cells. Although many of these approaches offer the advantageof real-time monitoring, the protocols required are laborious, oftenrequire cellular engineering, and have limited multiplexingcapabilities.

Newly developed microfluidic technologies and methods enable single-cellanalysis in a format that can be scaled to large numbers of cells.Microfluidic devices present a powerful platform for probing singlecells because the intrinsic length (1-100 μm) and volume scales(picoliters-nanoliters) are close to the size and volume of single cells(≈1 pL). The biggest advantage microfluidics offers is the ability tointegrate all processing steps into a single device, eliminating samplecontamination and product loss, which would preclude sensitive,reproducible, and quantitative single-cell analysis.

The 3 steps that must be integrated into a microdevice to performsingle-cell gene expression analysis are cell selection andlocalization, enzymatic reaction, and quantitative detection of theanalyte of interest. Although many microfluidic systems havedemonstrated 1 or 2 of these elements, the successful integration of all3 is extremely challenging. Early microfluidic systems successfullycoupled PCR chambers to capillary electrophoresis (CE) separationchannels. Recent integrated microsystems have demonstrated a significantincrease in detection sensitivity, the handling of crude samples, andmassive parallelism. Despite these advances, no integrated microfluidicdevice has successfully coupled all 3 steps into a single platform tomeasure changes in gene expression directly from single cells. Thefundamental hurdle has been the efficient transfer of analyte betweeneach nanoliter-processing step.

To address this challenge, we have developed an integrated microfluidicdevice with all of the necessary elements for single-cell geneexpression profiling and used it to perform a study of single-cell genesilencing (FIG. 20). Cells are functionalized with a 20-baseoligonucleotide on their surface to enable capture on a gold pad by DNAhybridization. Multiplex gene expression analysis from GAPDH mRNA andcontrol 18S rRNA is performed on 2 cell populations. The first cellpopulation consists of untreated Jurkat T lymphocyte cells grown undernormal conditions exhibiting homogenous high expression of both targetgenes (FIG. 20A). The second cell population is treated with siRNAdirected at GAPDH mRNA (FIG. 20B). The degree to which GAPDH mRNA issilenced in individual cells is probed relative to the 18S rRNA control.

The gene expression microdevice contains 4 independently addressablearrayed analysis systems on a 100-mm-diameter glass wafer (FIG. 21).Each of the identical microsystems contains 4 distinct regions that areintegrated to enable maximal transfer efficiency between processingsteps. The first region is a 3-valve pump for moving material from thesample inlet through the reactor region. In the reactor region, singlecells are captured, lysed, and the mRNA of interest is reversetranscribed and amplified by RT-PCR. The affinity capture regioncomprises a hold chamber that acts as a reservoir and a capture chamberwhere amplicons are immobilized, purified, and concentrated in anaffinity capture gel matrix. Finally, the system contains a CEseparation channel for the size-based separation and quantitation ofproducts.

The complete analysis from single-cell capture to CE separation anddetection is performed in <75 min as outlined in FIG. 22A. First, Jurkatcells are functionalized with a 20-base oligonucleotide (FIG. 22B).Jurkat cells are grown with a synthetic peracetylatedN-azidoacetylmannosamine (Ac₄ManNAz) sugar that is metabolized by thecell and results in the presentation of azido groups on the cells'surface. Phosphine-modified ssDNA is reacted with the azido group viaStaudinger ligation, generating a population of cells functionalizedwith ≈270,000 ssDNA molecules per cell.

In another embodiment, ssDNA molecules can be attached to the Jurkatcells using the present methods as described in Example 1 as follows:Immediately prior to modification, a sample of 5×10⁶ Jurkat cells arewashed with PBS buffer three times to remove any proteins from theculture medium. After the final rinse, additional PBS is added to bringthe volume to 5 mL (1×10⁶ cell/mL). The cell suspension is then reactedwith 1 mL of NHS-DNA (11.7 μM) solution synthesized and purified from 30μL of 5′-thiol ssDNA. The mixture is allowed to react at roomtemperature for 30 minutes, and is then washed three times with PBScontaining 1% FBS. The cells are then resuspended in 0.5 mL of PBScontaining 1% FBS

Inside the reactor, the complementary 20-base strand of thiol-modifiedcapture DNA is immobilized on a photolithographically defined 25-×25-μm²size-limiting gold pad with a gold-thiol linkage. Cells are flowed intothe reactor and immobilized on the gold pad via DNA hybridization. Thesize of the pad ensures that only 1 cell will bind. In previous reportswe have shown that the combination of metabolic engineering andDNA-based attachment leads to significantly less cellular activationthan antibody or lectin-based methods. Thus, although any attachmentmethod would be expected to have some effect on cell behavior, theDNA-based method minimizes these effects.

After washing the residual uncaptured cells out of the reactor, thecaptured cell is prepared for analysis (FIG. 22C). After a rapid 30-sfreeze-thaw lysis, the target mRNA is reverse-transcribed into a stablecDNA strand during a 15-min incubation at 42° C. Then, 30 cycles of PCRamplification are completed in the same 200-nL reactor in 25 min. AllRT-PCR products are then quantitatively transferred from the reactor tothe separation channel for size analysis. Amplified fragments andunreacted RT-PCR mixture are pumped from the reactor into the holdchamber and electrophoretically driven from the waste to the cathodereservoir. Fragments of interest with complementarity to the affinitycapture probe are quantitatively concentrated and immobilized at theentrance of the capture chamber, creating a purified capture plug. Theaffinity capture matrix comprises a linear polyacrylamide (LPA) gelcopolymerized with two 20-base oligonucleotide capture probescomplementary to the fragments of interest (20 μM). This capture processuses sequence-specific helix invasion to immobilize the dsDNA amplicons.The capture probes are complementary to sequences 23 and 47 bases fromthe end of the GAPDH and 18S rRNA amplicons, respectively. By placingthe probes internal to the priming sites, the capture gel also acts as apurification matrix to remove unreacted high-molarity FAM-labeledprimers. Finally, the purified and concentrated products are thermallyreleased at 80° C. from the affinity capture gel, electrophoreticallyseparated, and quantitated by confocal fluorescence detection (data notshown).

The integrated microfluidic gene expression analysis system yieldsquantitative data on the gene silencing of individual cells. Asexpected, the untreated Jurkat cells exhibit normal expression of GAPDHmRNA and 18S rRNA (FIG. 23A). The representative electropherogram shows2 strong peaks migrating at 160 s and 185 s for the 200-bp GAPDH and the247-bp 18S rRNA targets, respectively (FIG. 23). Moreover, the use ofthe capture matrix to immobilize the fragments of interest removes allunreacted primers from the separation, enabling us to look at both smalland large amplicons without interference. A single Jurkat cellelectroporated with siRNA directed at GAPDH mRNA produces only a singlepeak for 18S rRNA at 185 s. Single-cell experiments from 8 individualcells show expression of the GAPDH mRNA at 0, 5, 50, 1, 48, 0, 5, and 0%of untreated Jurkat cells (FIG. 23B). This analysis indicates thatsingle cells fall into populations with moderate (≈50%) or completesilencing (≈0%). These single-cell measurements differ fundamentallyfrom a bulk measurement performed on 50 Jurkat cells under the sameconditions where the expression of GAPDH is reduced to 21±4% (n=4) ofits original value. Thus, the ensemble average measured for genesilencing masks the stochastic diversity of individual cellularresponse. A control assay where no cell is captured on the pad exhibitsno products, verifying that there is no carryover contamination in thesystem. Similarly, a PCR control without reverse transcriptase shows noamplification, ensuring that the amplification template is RNA and notDNA.

To ensure that the variation in silencing behavior is not a simplefunction of the amount of siRNA introduced during the electroporationprocess, cells were treated with a fluorescently labeled siRNA. Cellsgrown under normal conditions showed an average uptake of theCy3-labeled siRNA of 17±2 relative fluorescence units (rfu) (Table S1)with 4 of 30 cells appearing at a slightly elevated level of 22 rfu. Ifelectroporation variability were the cause of the 2 cellularpopulations, we would expect 13% of the cells to be characterized ascompletely silenced and 87% as moderately silenced. The single-cell geneexpression analysis performed in the microfluidic device shows theopposite trend (FIG. 23B).

TABLE S1 Tabulated data from single-cell siRNA electroporationexperiment Cell no. Minimum Maximum Difference, rfu 1 26 41 15 2 25 4217 3 26 41 15 4 25 40 15 5 25 42 17 6 25 48 23 7 26 40 14 8 25 40 15 925 41 16 10 26 44 18 11 25 43 18 12 25 41 16 13 24 39 15 14 24 40 16 1524 42 18 16 24 46 22 17 24 42 18 18 24 42 18 19 25 42 17 20 25 44 19 2126 43 17 22 26 42 16 23 26 44 18 24 26 40 14 25 24 40 16 26 24 39 15 2724 46 22 28 21 37 16 29 22 44 22 30 23 40 17 Average: 17.2 SD: 2.4 Cellswere electroporated with Cy3-labeled siRNA and grown under normalconditions and Imaged with epifluorescence. Each cell is interragated,and the maximum pixel intensity value is recorded in addition, a minimumvalue is recorded from just outside each cell for normalization. Theaverage update is 17.2 ± 2.4 relative frequency units (rfu). The maximumupdate is 23 rfu, and the minimum is 15 rfu.

In addition, the GAPDH gene was sequenced to examine whether thepopulation of cells experiencing moderate silencing arises from aheterozygotic polymorphism in the siRNA-binding domain. The sequence wasfound to be without mutation. The sequencing result of GAPDH siRNAbinding sequencing showed that the expected sequence (5′-AAA GTT GTC ATGGAT GAC C-3′; SEQ ID NO:11) was found in the Jurkat cells, suggestingthat the 2 populations of cells are not a result a polymorphism in thebinding domain. Thus, the 2 populations of cells revealed here are not atrivial result of siRNA delivery, genetic variation, or cell viability.

The occurrence of 2 distinct cellular populations with different levelsof gene silencing has been detected by Liu Y P, Dambaeva S V, DovzhenkoO V, Garthwaite M A, Golos T G. Stable plasmid-based siRNA silencing ofgene expression in human embryonic stem cells. Stem Cells Dev. 2005;14:487-492. Measurements of enhanced green fluorescent proteinproduction in human embryonic stem cells (hESCs) have shown anall-or-nothing response to siRNA treatment, but the underlying mechanismwas not characterized. Our system demonstrates the unique ability toperform quantitative transcript analysis, revealing that although 80% ofthe cells exhibited the expected complete inhibition, 20% exhibited 50%inhibition. This suggests the presence of a genetic or phenotypicbistability or switch that controls the degradation of the siRNA, blocksits target binding, or inhibits transcript degradation. Of these onlythe latter 2 mechanisms would provide a nontrivial explanation for the50% inhibition level. A more exhaustive study is now called for toverify the biphasic expression trend observed here and to explore itsmechanistic origin. Because the fabrication of highly parallelstructures is one of the key advantages of our approach, scaling up to96-analyzers would provide the throughput necessary for this type ofstudy.

Our ability to perform 1-step RT-PCR amplification in our expressionmicrodevice with only 30 cycles of PCR in a single reactor is a directresult of efficient integration. First, the use of glass rather thanporous polymeric materials prevents product absorption. Second, the highthermal conductivity of glass enables rapid thermal cycling andincreased reaction efficiency. Third, the use of pneumatic valves andpumps allows for the efficient transfer of nanoliter bolus material.Finally, the affinity capture, purification, and concentration processenables the quantitative analysis of all generated products, a dramaticimprovement over the use of a traditional cross-injector or hydrodynamicpressure injector, which only permits a small portion (<1%) of theproducts to be analyzed. These advantageous attributes of our integrateddevice point the way to a wide variety of bioanalytical studies on theproperties and behavior of single cells.

Here, we have performed single-cell measurements on the variation ofmRNA knockdown as a result of siRNA treatment. This assay suggests aunique biphasic gene knockdown efficiency in individual cells that wasmasked by bulk measurements. Because the analysis step utilizes asize-based separation, the multiplex capabilities are determined by thenumber of products that can be generated and analyzed, suggesting thatthe expression of 5-10 targets could be studied in parallel. By couplingthis microdevice with laser capture microdissection, the heterogeneousnature of tumors could be investigated at the single-cell level. Itshould also be possible to perform a quantitative single-cell analysisof the effects of siRNA treatment on expression in hESCs when Oct4 mRNAtargeting is used to trigger differentiation into trophoblast-likecells. Moreover, based on our previous detection of <11 mRNA moleculesper reactor, our microfluidic device may ultimately enable studies ofexpression from individual cells at the single-transcript level, onceimproved product capture, purification, and injection processes arefully enabled and integrated. Overall, our approach offers many excitingprospects for revealing the stochastic variation in gene expression thatunderlies the ensemble average.

Materials and Methods. Bioprocessor Fabrication.

The fabrication protocol is similar to that used in previous nucleicacid amplification microdevices, Toriello N M, Liu C N, Mathies R A.Multichannel reverse transcription-polymerase chain reaction microdevicefor rapid gene expression and biomarker analysis. Anal Chem. 2006;78:7997-8003 and hereby incorporated by reference. Briefly, to form thepneumatic manifold wafer, valve seats and actuation channels werephotolithographically defined and etched to a depth of 38 μm on a0.5-mm-thick borofloat 100-mm glass wafer. Valve actuation access holeswere drilled, and the manifold was diced into reusable 9-mm×6-cm strips.Removable polydimethylsiloxane (PDMS) elastomer valves were formed byactivating both sides of the 254-μm PDMS membrane with a UV ozonecleaner for 1.5 min to improve PDMS-glass bonding and then sandwichingthe membrane between the manifold and the bonded channel wafers.

The reactor/channel wafer was fabricated on a 0.5-mm-thick borofloatglass wafer. Fluidic channels for pumping were photolithographicallydefined on the front side and etched to a depth of 38 μm. Reaction,hold, and capture chambers along with separation channels werephotolithographically defined on the back side and etched to a depth of20 μm. Electrophoresis reservoirs, resistance temperature detection(RTD) access holes, and valve via holes were diamond drilled. To formthe RTD wafer, a 0.5-mm-thick borofloat glass wafer sputter depositedwith 200 Å of Ti and 2,000 Å of Pt (Ti/Pt; UHV Sputtering) wasphotolithographically patterned and etched with 90° C. aqua regia toform the 30-μm-wide RTD elements and 300-μm-wide leads. The drilledreactor/channel wafer was aligned and thermally bonded to the RTD waferby using a programmable vacuum furnace at 655° C. for 6 hours.

To form the removable modular heater, a 0.5-mm-thick borofloat glasswafer was sputter-deposited with 2,200 Å of Ti/Pt. Heater leads wereformed by electroplating 6 μm of gold onto photolithographically definedareas. Ti/Pt serpentine resistive heater elements connecting the goldleads were formed by anisotropically etching photolithographicallyexposed Ti/Pt in an ion mill.

Jurkat Cell Preparation.

T lymphocyte Jurkat cells were cultured in 50-mL flasks (Nalge-NuncInternational) for 48 h in 10 mL of medium (RPMI medium 1640;Invitrogen) containing 1% penicillin/streptomycin (1% P/S; Invitrogen)and 25 μM Ac₄ManNAz resulting in the display of N-azidoacetylsialicacids on the cell surface glycans. For the first 24 h of growth, thecells were cultured with 10% FBS (JR Scientific). The Jurkat cells werewashed and incubated in serum-depleted medium containing 25 μM Ac₄ManNAzfor an additional 24 h for synchronization. Fresh DNA-functionalizedJurkat cells were prepared 1 h before the analysis. Cells were washedtwice with 5 mL of PBS (Ambion) containing 1% FBS and reacted with 125μM phosphine-modified ssDNA (5′-phos-GTA ACG ATC CAG CTG TCA CT-3′; SEQID NO:12) in 1% FBS/PBS for 1 h at 37° C. The cells were then rinsed 3times with 5 mL of 1% FBS/PBS solution before introduction into themicrofluidic device.

siRNA Treatment.

For gene-silencing studies, 150,000 Jurkat cells were electroporatedwith 2.5 μg of double-stranded GAPDH siRNA (sense, 5′-GGU CAU CCA UGACAA CUU UdTdT-3′ (SEQ ID NO:13); Ambion). Cells were suspended in 75 μLof siPORT electroporation buffer (AM1629; Ambion) and a single pulse isperformed in a 1-mm cuvette (Bio-Rad) for 250 μs at 250 V. Cells werethen grown and prepared in the same manner as described in the Jurkatcell preparation section above. For negative control studies, cells wereelectroporated with 150 pmol of Cy3-labeled siRNA that does not bind tomRNA.

RT-PCR Mixture.

Multiplex RNA RT-PCR was performed on GAPDH and 18S rRNA transcriptsdirectly from Jurkat cells. A 25-μL, RT reaction mixture comprises aCell-to-cDNA II kit [4 units of Moloney murine leukemia virus (Mo-MLV)reverse transcriptase, 0.4 unit of RNase inhibitor, 0.1 μM dNTPs, 1×RTbuffer (Ambion)], 0.08 unit of platinum Taq polymerase (Invitrogen),along with 800 nM forward and reverse primers for the GAPDH gene and 20nM forward and reverse primer for the 18S rRNA target. The GAPDH forward(5′-AGG GCT GCT TTT AAC TCT GG-3′; SEQ ID NO:14) and reverse (5′-FAM-TTGATT TTG GAG GGA TCT CG-3′; SEQ ID NO:15) primers generate a 200-bpamplicon. The 18S rRNA forward (5′-CGG CTA CCA CAT CCA AGG AAG-3′; SEQID NO:16) and reverse (5′-FAM-CGC TCC CAA GAT CCA ACT AC-3′; SEQ IDNO:17) primers generate a 247-bp amplicon. Controls without RT andwithout template were performed on the microdevice by removing theMo-MLV RT and Jurkat cells from the reaction mixture, respectively.

Matrix Synthesis.

A DNA affinity capture gel is synthesized by copolymerizing LPA with 25′-acrydite-modified capture oligonucleotides. The affinity capturematrix is synthesized at 4° C. by sparging a 2-mL solution containing 6%wt/vol acrylamide, 1×TTE, and 40 nmol of the 2 acrydite-modifiedoligonucleotides (IDT) for 2 h with argon followed by the addition of0.015% wt/vol ammonium persulfate (APS; Fisher Scientific) andtetramethylethylenediamine (TEMED; Fisher Scientific). The affinitycapture matrix contains capture probes for GAPDH (5′-Acry-ATC CCA TCACCA TCT TCC AG-3′ (SEQ ID NO:18), T_(M)=54.2; 50 mM monovalent salt, 20μM) and 18S rRNA (5′-Acry-GCA GCC GCG GTA ATT CCA GC-3′ (SEQ ID NO:19),T_(M)=61.9; 50 mM monovalent salt, 20 μM). The GAPDH captureoligonucleotide is complementary to a 20-base sequence in the 200-bpamplicon, 23 bases from the 5′ FAM-labeled terminus. The 18S rRNAcapture oligonucleotide is complementary to a 20-base sequence in the247-bp amplicon, 60 bases from the 5′ FAM-labeled terminus.

Reactor Preparation.

The glass surface is derivatized with polydimethylacrylamide (PDMA) byusing a modified Hjerten coating protocol to prevent nonspecific celladhesion. First, the reactors glass surface is deprotonated byincubating with 1 M NaOH for 1 h. The NaOH solution is replaced with a0.6% (vol/vol) (γ-methacryloxypropyl)trimethoxysilane solution (γ,Sigma) in 3.5 pH H₂O. The bifunctional γ-solution prepares the glasssurface for acrylamide polymer nucleation. During γ-solution incubation,250 μL of dimethylacrylamide is dissolved in 4.75 mL of H₂O and spargedwith Ar for 1 h. After Ar sparging, 100 μL of isopropyl alcohol (IPA),20 μL of TEMED, and 25 μL of 10% (vol/vol) APS were sequentially addedto the acrylamide solution to form linear PDMA. The γ-solution isremoved from the channel, and PDMA solution incubates in the channel for1 h. The channel is then rinsed and dried with acetonitrile.

Next, the photolithographically defined 25-μm×25-μm gold pad in thecenter of the reaction chamber is functionalized with ssDNA byincubating for 1 h with Tris(2-carboxyethyl)phosphine (TCEP, 200 μM;Invitrogen) deprotected thiol-DNA (5′-thiol-AGT GAC AGC TGG ATC GTTAC-3′ (SEQ ID NO:20), 20 μM). The chamber is then rinsed and dried toremove unbound DNA.

Gel Loading Sequence.

The device is prepared for affinity capture and separation by treatingthe separation channels, hold chambers, and capture chambers with adynamic coating diluted in methanol for 1 min (1:1; DEH-100; The GelCompany) to suppress electroosmotic flow. Multiplex affinity capturematrix (20 μM, 6%, yellow) is loaded from each cathode (C) reservoir upto the separation channel cross at room temperature. Separation matrix(red) is then loaded from the central anode (A) past the capture chamberto the sample load cross. With all of the valves opened, the rest of thesystem is hydrated by adding 3 μL of RNase-free water at the sampleports and applying a vacuum at the waste (W) reservoirs. The microdeviceis then placed on a 44° C. temperature-controlled stage. An additional 2μL of water is flushed through the system to remove thermally expandedgel from the sample load cross.

Bioprocessor Operation.

As depicted in FIG. 22, the microdevice operation begins by preparingthe reaction chamber for cell capture. Cell modified to contain ssDNA ontheir surface were suspended in the reaction mixture and drawn into thereaction by vacuum. When a cell flows over the gold cell capture pad,DNA hybridization occurs between the ssDNA on the surface of the celland the complementary ssDNA immobilized on the gold pad. To maximizecapture efficiency, DNA-mediated cell capture was performed for 15 min.The uncaptured cells were washed out of the system and removed at thewaste port, and the valves were closed for thermal cycling. During thisstudy, all 4 reactors were used in parallel. This enabled simultaneousanalysis from 4 individual cells. After capture, the morphology of eachcell is recorded to ensure that it has remained viable. The total datacollection time for the single-cell studies was 1 month.

Freeze-thaw lysis and 1-step RT-PCR thermal cycling were performed in asingle 200-nL reactor. A piece of dry ice was placed over the reactionchambers of all 4 reactors for 30 s to freeze-thaw lyse the capturedJurkat cell. Freeze-thaw lysis was used in this 1-step RT-PCR to preventearly unwanted activation of the Hot Start Taq polymerase, to preventdenaturation of the reverse transcriptase enzyme, and to minimize RNAdegradation by RNases. Next, a linear 15-min cDNA synthesis from thecells' RNA was performed at 42° C. by using primers complementary to theRNA transcripts of interest (GAPDH and 18S rRNA). After cDNA synthesis,the Mo-MLV RT was denatured, and the platinum Taq polymerase wasactivated at 95° C. for 60 s followed by 30 cycles of PCR at 95° C. for5 s, 47° C. for 20 s, and 72° C. for 25 s. Because of the rapid heatingand cooling rates (>15° C. s⁻¹), each cycle of PCR is completed in 50 s,and the total reaction time is 46 min.

After thermal cycling, affinity capture, purification and concentrationof the products of interest were performed. The reactor contents werepumped into the hold chamber by using a 5-step pump cycle. A 350-msactuation was used with each step, resulting in a 30-nL stroke volume. A23-s delay was used between each pump cycle to allow sufficient time forthe analyte to migrate into the capture region and to prevent analyteaccumulation in the hold chamber. A constant 100-V/cm field between thewaste (W) and cathode (C) reservoirs electrophoretically drives theanalyte toward the capture chamber. Analytes complementary to thecapture probe were hybridized at the entrance of the capture chamber,creating a sample plug. The electric field between the waste and cathodewas maintained until residual PCR reactants (excess primer, salts, andbuffer) were washed into the cathode reservoir thus resulting in apurified amplicon sample plug. Thirty pump cycles were used resulting ina total capture and wash time of 12.2 min. After the capture process wascompleted, the temperature of the entire device was raised to 80° C. tothermally release the captured DNA fragments thermally from the affinitycapture gel, and the sample was separated with a field of 150 V/cmbetween the cathode and the anode. The electrophoretically separatedFAM-labeled products from all 4 lanes were detected by usinglaser-induced fluorescence with the Berkeley rotary confocal scanner(Shi Y N, et al. Radial capillary array electrophoresis microplate andscanner for high-performance nucleic acid analysis. Anal Chem. 1999;71:5354-5361). The entire capture and release process was performed on atemperature-controlled stage on the scanner to prevent thermalgradients.

Example 7 Patterning of Single Algal or Bacterial Cells for Bioreactoror Fuel Cell

Using the principles of the invention as applied to oligonucleotideattachment on non-animal cells through an aldehyde or ketone on acarbohydrate, hydrogen production efficiency can be studied andoptimized. The oligonucleotide capture and attachment to a device allowthe creation of patterned cells and enzymes on a chip. In the system,conversion of hydrogen gas to electricity measures hydrogen production.Measuring direct hydrogen production via current generation isadvantageous because of potentially higher sensitivity to hydrogenproduction compared to conventional measurements (see GC readings ofculture headspace). The advantage of the invention to such studies andactuation include the attachment and control implicit with preciseplacement of the cells on the device, that the fuel cell can then bereusable, and also higher efficiency in utilization of solar and carbonenergy For example, a self-sustaining fuel cell device can beconstructed using patterned layers of different photosynthetic cells,where the cells absorb sunlight in non-overlapping parts of the solarspectrum, and also incorporating alternating layer(s) of nitrogen-fixingorganisms which feed on and utilize the biomass created by thephotosynthetic cells in adjacent layers. When exhausted, the fuel cellhaving the cells attached by oligonucleotide hybridization can beheated, the oligonucleotides dehybridize, and dead cells wash away. Thesurface is rinsed over with fresh modified cells which reattach tocomplementary oligonucleotides on the device surface. However, sol geldevices having solgel embedded cells are discarded and cannot be reusedin the same manner, but this turnover may be appropriate to theirtargeted application.

Optimized bacterial and algae cell patterning is accomplished usingsuspended cells in 5 mM NaIO₄ in DPBS for 20 minutes, at 37 degreescentigrade. After rinse with DPBS, the cells are resuspended in 10 mManiline in pH 6.0 MOPS buffer and 35 μM I-linker-DNA (from IDT, I linkeris a hydrazide linker) for 2 hours. The cells are rinsed, incubated orcentrifuged (1% FBS) to complementary patterned DNA for several minutes.In addition, to other advantages already stated, when usingoligonucleotide captured cells and exposing them to sunlight as a sourceof energy for bioreactor, bioactivity, metabolic, and fuel cellelectricity production, and other such similar uses, the inventionprovides the opportunity to arrange the cells in a monolayer or multiplelayers, each cell having direct access to the solar fuel source. Inturn, this allows the opportunity to employ lower intensity light.

Other experiments and uses of the system are to optimize a design for aproton exchange membrane hydrogen fuel cell on a chip. Additionally,porous polymer must be used to prevent poisoning of theplatinum/palladium by nitrogenous bases and cell waste. To determinehydrogen output efficiencies in permutations of such a system, currentgenerated per unit time is measured.

Hydrogen output efficiency of different photosynthetic andnon-photosynthetic microorganism combinations can be studied on the pathtowards increasing system efficiency. Parameters such as the patterns ofthe organisms on the device, light exposure, and specific combinationsof organisms in patterns can be varied to optimize efficiency. Forexample, a three organism hydrogen production system proposed by Melislab (two photoautotrophs that absorb different regions of solarradiation and one heterotroph that takes the biomass created by thephotosynthesis and converts it to small organic acids which thephotosynths need for autotrophic growth) can be adopted. Proposedorganisms include but are not limited to, Photosynthetic: C. reinhardtii(algae), Synechocystis PCC 6803 (cyanobacteria), R. rubrum (gramnegative anaerobe); Heterotrophs: C. pasteurianum (nitrogen fixer),Azotobacter sp. (proposed to have the highest respiration rate of anyorganism). (see Melis et al, cited elsewhere herein).

In another embodiment, the fuel cell device comprising an open aircathode instead of solution-based reduction at the cathode, and an anodepatterned with bacteria directly on the anode. This will be done bycoating the anode with LPA (linear polyacrylamide) modified withstreptavidin (available through invitrogen). The coating will also actto protect the Pt from poisoning by nitrogenous waste from the cells.5′-biotin modified DNA will be patterned on the LPA coated electrodesusing aluminum liftoff lithography patterning. So, the glass electrodebase will be fabricated first then the DNA will be patterned on theelectrodes, finally the cells will be patterned on the electrode and thePDMS top will be bonded on. There will constantly be a very slow flow ofmedia into the cell because hydrogen production is greatest during logphase growth and when nutrients are plentiful. The device will beilluminated and the photosynthetic hydrogen production can be measuredfor example, by a voltage drop over a resistor with some sort of a biasplaced on the cell.

In a fuel cell device, the hydrogen gas will nucleate on the Ptelectrode and as with most fuel cell designs, the electrons will beremoved, immediately converting the hydrogen produced by the cells intoelectricity.

Another interesting aspect of patterning is using the DNA to pattern thecells on top of each other for use in a fuel cell device. Referring nowto FIG. 30, multiple cells can be patterned on top of each other on aDNA microarray. Each cell is labeled with a different fluorophor, exceptthe cyanobacteria Synechocystis, which auto-fluoresces red. A schematicof the cells and what fluorophor they are labeled is shown in FIG. 30.The advantage is that the spatial orientation of the oxygen absorbingcell (A. vinelandii) sandwiched between the two photosynthetic cellswill be potentially useful in keeping oxygen concentrations low in thelocal vicinity of the photosynthetic cells. Since photosynthesisgenerates oxygen (at least in the case of algae and cyanobacteria) as abyproduct and the enzymes which make hydrogen are inhibited by oxygen,having the photosynthetic cells tethered to a cell with an incrediblyhigh metabolism means that local oxygen concentration should always below and hydrogen production will be sustained. Currently, Synechocystisdoes not do well at long term H₂ production due to the oxygen inhibitionof its hydrogenase enzymes. The presently anticipated experiments shouldshow that cultivating the cells together can be done (such as A.vinelandii and Synechocystis co-cultures, A vinelandii and R rubrumco-cultures, or all three cultured together) and show that thesecultures have amiable hydrogen evolution and lower concentrations ofoxygen present than if the cells were cultured alone.

O₂ and H₂ gas concentration can be measured using gas chromatography.Upon building the device and it can be tested using one cell type (e.g.,R. rubrum) to show it can work as a hydrogen fuel cell. The finalexperiment will be investigating how two and three dimensionalpatterning will effect the electrical output (synonymous with H₂production) of the cell when using 2 or more cell type combinations.

The strands used to modify each cell do not complement any other strandson the other cells or glass to insure that the cells are patternedcorrectly. The same oligonucleotides used in the previous examples(M1/C1, M2/C2, M3/C3) and two other complementary sequences Z2 (5′CACACACACACACACACACA 3′; SEQ ID NO:21) and zc2 (5′ TGTGTGTGTGTGTGTGTGTG3′; SEQ ID NO:22) can be used to pattern these cells onto the glasssubstrate. The cells are patterned one layer at a time. The cells areoxidized in 5 mM sodium periodate and then incubated in ˜35 μMhydrazide-DNA at pH 6 in MOPS (N-morpholino propane sulfonic acid)buffer (note that MOPS is being used in this experiment outside itsnormal buffering range). After washing the cells, they are reversiblybound to a PDMS well on the glass substrate which has been previouslymodified with DNA. The well is filled with one cell solution,centrifuged at 3000 rpm for 5 min, rinsed in PBS and repeated with thenext two or three cell types sequentially. The DNA should notdehybridize under these conditions allowing the cells to be patterned asshown in FIG. 30. The cell types chosen are not limited to those shown,but multiple layers of patterned cells can be added using the presentmethods. For example, referring now to FIG. 30, the glass can bemodified with M2 oligonucleotide. The layer of Synechocystis cells areattached to the glass substrate through the C1 oligonucleotide. ThisSynechocystis layer is then modified with the C2 oligonucleotide. The A.vinelandii cells are modified with M2 oligonucleotide and are attachedto the first Synechocystis layer through M2/C2 hybridization. Layer twoof A. vinelandii cells are modified with M2 and C3, and the third layerof R. rubrum cells are modified with M3 and which then hybridize to theM2 oligonucleotide and link the third layer to the second layer ofcells.

Patterning should be carried out under sterile conditions and thenplacing the pattern in media in which all of the cells can grow and in avial with a small amount of head space. H₂ production could be monitoredby taking samples from this headspace over time and monitoring activity.The system will require a medium in which all three or two or howevermany cell types are patterned can live and a light source, such assunlight or a fluorescent or tungsten bulb is sufficient. In the devicea slow flow of media is provided into and out of the chamber where thecells are patterned on the electrode. No gas will be needed to maintainthe system. However, nitrogen gas will be used to sparge the system whenstarting the fuel cell in order to create an anaerobic environment.

Any medium can be used which contains the same nutrient concentrationsnecessary in each cell's individual medium but in the volume of onemedium. For instance, Synechocystis uses a medium called BG11 and A.vinelandii lives in Burk's medium so a cocultivation medium containingall of the nutrient ingredients in 1 L of BG11 and 1 L Burk's medium butin a single volume (1 L) is made, thereby providing a higherconcentration of nutrients per liter compared to the individual medias.R. rubrum uses a medium ORMERODs so when a culture for all threeorganisms will have all of the ingredients (minus the phosphate forbuffering) for 1 L of each culture in 1 L of cocultivation medium makinga three media in one high nutrient media. The phosphate buffer recipefrom the Burk's medium can be used to optimize the phosphateconcentration.

The device would work for powering things which are outside constantly.When the cells die the device is heated up, the DNA dehybridizes, thedead cells wash out, and fresh cells are washed in to replace the oldones. The device could also act as a battery or an addition to thepowergrid. In theory, many of the devices could be wired in series topotentially power vehicles or could act synonymously with Si solarpanels.

Example 8 AFM Patterning

The forces governing cell-cell adhesion are vitally important to manybiological processes, including cell differentiation, tissue growth,tumorigenesis, and proper functioning of the vertebrate immune response.The strengths of these interactions are typically characterized throughthe attachment of single living cells to probes that are capable offorce measurement, such as suction micropipettes. More recently, opticaltweezers have been applied to capture single cells and to measure theseforces with high accuracy, but this technique is limited to applyingforces in the piconewton range. Atomic force microscopy (AFM) providesan attractive alternative to these methods, because it is capable ofquantifying forces in the piconewton to nanonewton range, and thistechnique has indeed been used to measure the mechanical properties oflive single cells and to study adhesion forces at the single-cell level.Several fundamental adhesion measurements have been achieved by coatingAFM cantilevers with fibronectin or lectins that bind to carbohydratemoieties on the cell surface, but especially in the latter case thecell-binding molecules themselves have been reported to have a degree ofcytotoxicity that can influence the cellular properties being evaluated.Thus, while these studies highlight the utility of AFM for themeasurement of cell receptor-ligand interactions, an expanded set ofcantilever attachment methods will be needed for the study of cell-cellinteractions over widely varying time scales.

To address this need, we have compared three biomolecule-mediatedmethods for the attachment of live cells to AFM cantilevers, with anemphasis on the cell viability, adhesion strength, and probe reuse thateach technique can achieve. These studies have indicated that cellattachment through the use of complementary DNA strands has the leastinfluence on viability and does not appear to activate cell signalingpathways. This method also offers overall superior adhesion strength,but this parameter can be attenuated to allow cells to be transferredfrom one surface to another. We were able to demonstrate this concept bypicking up free cells and placing them in exact positions on a substratebearing DNA strands with longer complementary regions. This “dip-pen”live-cell patterning demonstrates the reusability of the DNA-mediatedcell adhesion method and could prove useful for the construction ofcomplex mixtures of cells with well-defined spatial relationships.

To allow the comparison of several attachment strategies, threedifferent biomolecules (DNA, concanavalin A (ConA), and an antibody)were attached to silicon nitride AFM cantilevers for cell anchoring. Forall attachment methods, the thin layer of silicon oxide on the workingsurface was covered with aldehyde groups as outlined in FIG. 16a . Thesurfaces produced using these steps were characterized by contact-anglemeasurements.

Amine-functionalized DNA was attached to the aldehyde groups throughreductive amination (FIG. 16b ). First, the aldehyde-coated cantileverwas immersed in an amine-functionalized single-strand DNA (ssDNA)solution and then heated to promote imine formation. After cooling toroom temperature, an aqueous solution of sodium borohydride was used toreduce the imines to nonhydrolyzable amine linkages. This step alsoserved to reduce any unreacted aldehyde functional groups to alcohols.By coupling 5′-amine-functionalized DNA strands bearing fluoresceinisothiocyanate (FITC) at the 3′ end, the presence of the strands couldbe verified by fluorescence imaging.

In previous efforts, proteins have been attached to AFM tips throughnonspecific adsorption and through glutaraldehyde crosslinking to aminegroups introduced on the tip surface. To afford more well-definedlinkages (and thus realize more homogeneous cell attachment), we choseinstead to use the simple reductive amination strategy that was used forthe amino-DNA strands. Surface lysine residues on ConA and anti-humanCD3 antibodies (anti-CD3) were reacted with the aldehyde functionalgroups on the cantilever surfaces (FIG. 16b ), but a lower concentrationof reducing agent (66 μM) was used to minimize the reduction ofdisulfide bonds that are required to maintain protein tertiarystructure. The concentrations of the proteins (20 μM ConA and 6 μManti-CD3) used in the reactions are easily achieved using commerciallyavailable samples. As described above for DNA, FTIC-labeled ConA andanti-CD3 samples were used in some experiments to verify biomoleculeattachment using fluorescence microscopy. Similar levels of fluorescencewere detected for each.

For the covalent attachment of DNA, lectins, and antibodies tocantilevers, the following methods were used. For DNA-mediated celladhesion studies, a complementary oligonucleotide sequence pair (A/A′)was designed. The sequence identities were as follows:

A: (SEQ ID NO: 23) 5′-TCA TAC GAC TCA CTC TAG GG-3′ A′: (SEQ ID NO: 24)5′-CCC TAG AGT GAG TCG TAT GA-3′

An aldehyde-coated cantilever (MLCT-NONM) was immersed into a 20 μMsolution of 5′-amine functionalized ssDNA in 3× saline/sodium citratebuffer (45 mM sodium citrate, 450 mM NaCl, pH 7.0) for 15 min, heated inan oven at 100° C. for 30 min, and then washed with 0.2% SDS solutionand distilled water (1 min each). The resulting cantilever was soaked ina fresh solution of 0.1 g of NaBH4 in 10 mL of ethanol and 30 mL of PBSsolution for 15 min, and then it was washed with 0.2% SDS solution andwater (1 min each). The cantilever was dried under N2 and stored in alow moisture environment until use. The ssDNA coated cantilever wascharacterized by coupling 3′-FITC-labeled 5′-amino ssDNA (A strand) tothe aldehyde-coated cantilever surface, followed by imaging with afluorescence microscope.

Concanavalin A and anti-CD3 IgG monoclonal antibodies were also coupledto an aldehyde-coated cantilever (MLCT-AUNM) surface by a reductiveanimation procedure. An aldehyde-coated cantilever was exposed to a 20μM (Con A) or 1 mg/mL (Anti-CD3) solution of the protein in pH 7.0 PBSbuffer solution containing 66 μM NaBH4 in a humid chamber for 2 h. Thecantilever was then washed with excess PBS and water, and stored in purePBS solution at 4° C. until use.

To prepare live cells bearing ssDNA on their surfaces, we firstintroduced azide functional groups into glycoproteins embedded in theplasma membrane, as previously described [Zabzdyr J L, Lillard S J.Measurement of single-cell gene expression using capillaryelectrophoresis. Anal Chem. 2001; 73:5771-5775]. PeracetylatedN-α-azidoacetylmannosamine (Ac₄ManNAz) was added to cells, which thenmetabolized and displayed the azide on their surfaces (FIG. 16c ).Triarylphosphine-modified ssDNA was prepared through the reaction of5′-amine-modified ssDNA with a phosphine pentafluorophenyl (PFP) ester.This reagent was then used to label the cell-surface azide groupsthrough Staudinger ligation, yielding stable amide linkages. Flowcytometry experiments have previously verified the ability ofphosphine-DNA conjugates to undergo ligation to azide-modified cellsurfaces. Although many cell types would be expected to be compatiblewith this system (and have been explored previously using the DNA-basedadhesion method), non-adherent Jurkat cells were chosen for thesestudies, because they do not secrete their own extracellular matrix.Thus, all cell adhesion events arise solely from the biomolecules ontheir surfaces

In other studies, to prepare live cells bearing ssDNA on their surfaces,the general protocol for the attachment of DNA strands to cells can becarried out. Immediately prior to modification, a sample of 5×10⁶ Jurkatcells are washed with PBS buffer three times to remove any proteins fromthe culture medium. After the final rinse, additional PBS is added tobring the volume to 5 mL (1×10⁶ cell/mL). The cell suspension is thenreacted with 1 mL of NHS-DNA (11.7 μM) solution synthesized and purifiedfrom 30 μL of 5′-thiol ssDNA (C2 sequence). The mixture is allowed toreact at room temperature for 30 minutes, and then washed three timeswith PBS containing 1% FBS. The cells are then resuspended in 0.5 mL ofPBS containing 1% FBS.

The effects of the adhesion molecules on the viability of the cells wereassessed using two different methods. First, suspensions of unmodifiedJurkat cells were supplemented with ConA or anti-CD3 antibodies, and thesolutions of DNA-coated cells were supplemented with the complementarysequence. FIG. 17a shows the growth curves of the resulting cells over athree-day period. The propagation of the DNA-modified cells was the sameas that of unmodified cells, but the anti-CD3-treated cells showeddelayed growth. ConA-coated cells aggregated and were no longer aliveafter 12 h. Cell morphology changes induced by soluble biomolecules.Jurkat cells were grown in normal media (Control), DNA-modified Jurkatcells were grown in the presence of 2 μM DNA, and unmodified Jurkatcells were grown in the presence of 2 μM ConA or 0.1 mg/mL Anti-CD3. Theresulting cells were examined under a light microscope after a period of12 h. The appearance of the cells was largely unchanged in the presenceof DNA, but cells grown in the presence of ConA and Anti-CD3 exhibitedaggregation and other morphological changes.

As a second comparison method, the three cell-adhesion molecules werecoated onto commercially available aldehyde-coated glass slides usingthe same reductive amination procedures outlined above. By visualinspection, all three surfaces were able to achieve efficient cellbinding (FIG. 17b ), but only the DNA-conjugated cells appearedmorphologically unchanged after 48 h. The ConA- and anti-CD3-immobilizedcells exhibited significant changes during this time period, likelyowing to crosslinking of their surface receptors. The viability of thesurface-immobilized cells was determined after 24 and 48 h using annexinV and propidium iodide (PI) staining. For the DNA-immobilized cells, thelow percentage of apoptotic and necrotic cells was similar to that ofunmodified cells (FIG. 17c ). However, the ConA and anti-CD3 immobilizedcells showed significantly higher numbers of apoptotic cells compared tothe control samples. Thus, the DNA molecules appear only to hybridizewith their complementary partners and should have much less potential todisturb the overall physiology of the cells in force measurementexperiments.

Live cells were readily captured by AFM tips bearing all three of thebiomolecules. This capture was accomplished simply by touching the cellmembrane with the cantilevers, with contact times as short as fiveseconds resulting in the transfer of the cells to the AFM tips. No cellswere captured by tips lacking the appropriate biomolecules.

Our assay to determine the strength of cantilever attachment wasdesigned such that cell-cantilever adhesions were fewer in number, andtherefore weaker overall, than DNA-based adhesions between a cell andthe complementarily functionalized glass slide. Owing to thisarrangement the cell-cantilever interaction would be expected to rupturefirst, yielding the strength of the interaction that a relatively lowconcentration of biomolecules can achieve. Rupture of thecell-cantilever interaction before the cell-surface interaction wasverified by visual observation during experiments. The force ofde-adhesion was measured for each attachment method using two differentretraction rates and two different contact forces (FIG. 18a ). Themeasured force of de-adhesion increased with contact force andretraction rate across all attachment methods, as predicted by the Bellmodel. The ConA attachment method yielded zero-force attachment eventsin 12% of the de-adhesion measurements. Such events were not observed inthe DNA and antibody cases

A significant spread of forces was observed for all three attachmentmethods; however, under all experimental parameters, the DNA methoddisplayed the strongest average adhesion, followed by antibodyattachment, then ConA (FIG. 18b ). As a control experiment, we alsodemonstrated that the capture efficiency of ConA and anti-CD3 is notaffected by the presence of DNA strands introduced on the cell surface.It should be noted that the overall de-adhesion forces determined foreach attachment strategy depend on both the number of linkages and theretraction rates and therefore do not reflect the absolute strengths ofthe individual biomolecular interactions. For comparison, the forcerequired to separate a typical 20 bp DNA duplex has been previouslydetermined to be 38-50 pN, suggesting that in our experiments roughly20-25 individual linkages are made between the cell and the cantileverif the interaction strengths are assumed to be simply additive, thoughmultiple parallel bonds can exhibit more complicated scaling behavior.Similar reasoning would suggest that about ten ConA-mannose interactions(at 47 pN each) and 12 antibody-antigen interactions (at 49 pN each) areinvolved. Experiments to determine the number of linkages involved ineach adhesion event are in progress to determine these effects moreaccurately. Nevertheless, our current results show that the DNAhybridization method leads to the most robust attachment under typicalpreparation conditions, even though the strength of each individuallinkage is likely to be less than that of the other biomolecules.

The strength of the cell-cantilever interaction can be tuned by varyingthe number of interacting strands and the length of the complementaryregions, and the reversibility of DNA hybridization also allows the tipsto be used many times. Both of these advantages allowed us to use AFMtips to arrange cells one at a time into patterns. In a recent report,it was shown that individual DNA strands could be moved from onelocation to another on a printed substrate, allowing small-molecule dyesto be printed in a similar fashion.

To do this, a 5 μM solution of a shorter DNA strand (13 bases) wasapplied to the cantilever, and an 80 μM solution of a longer strand (20bases) was coupled to the glass slide. DNA-coated Jurkat cells wereincubated in CO₂-independent media and applied to the uncoated side ofglass slide under an AFM instrument. The cantilever was then loweredinto contact with the cell for ten seconds with a contact force of 400pN. The cantilever was then retracted, and cell attachment to thecantilever was confirmed visually. The attached cells were then moved tothe DNA-coated side with maximum rate of 1 mm s⁻¹. The cantilever waslowered into contact with the slide, and the cell was allowed tointeract with the substrate for ten seconds with a 400 pN contact force.The cantilever was then retracted, whereupon the cell remained attachedto the glass slide. By applying this printing method, cells can be givenan (x,y) coordinate to position them precisely on a 2D substrate (FIG.19). The cells were found to remain viable after patterning.

In summary, we have described the development of a versatile DNA-basedadhesion method for the study of cell-cell interactions by AFM. The keyadvantages of this platform include the reusability of the tip, thetunability of the interaction strength, and the use of well-definedchemical linkages. Of the three biomolecule-based attachment strategiesthat were used, the DNA method proved superior in terms of cellviability after attachment. The use of AFM to form accurate andprogrammable patterns of individual cells provides a useful tool tounderstand the influence of neighboring interactions on celldifferentiation and regulation. In a previous report, we have shown thatcomplex patterns can be prepared through the self-assembly of DNA-coatedcells on surfaces printed with complementary oligonucleotides. The AFMdip-pen method described herein provides a useful complement to thistechnique that can achieve the higher resolution needed to create andinterrogate clusters consisting of multiple cell types. We are currentlyusing this method to elucidate fundamental adhesion mechanisms involvedin cancer metastasis, immune synapse formation, and cell-cellcommunication.

Example 9 Viral Capsids

Modification of Viral Capsids for Multivalent Targeted DeliveryVehicles.

Multivalent and targeted delivery vehicles offer great promise for drugadministration and diagnostic imaging. A number of core scaffolds,including polymers, dendrimers, inorganic nanoparticles, and liposomes,have been used with considerable success in these applications. In termsof biomolecule-based vectors, engineered heat shock cages and viralcapsids have also been developed to house drug molecules on theirinterior. For each of these carrier types, a critically importantconsideration is the installation of receptor-binding groups that enablethe selective association of the carriers with targeted tissue types.The most common molecular strategies for this purpose have involvedfolic acid, cobalamin, carbohydrates, peptides and antibodies, andnucleic acid aptamers. The rich chemical diversity of these molecules,added to the desire to attach multiple copies of each to scaffolds ofvarying composition, calls for chemical reactions that are exquisitelyfunctional-group tolerant and proceed under physiological conditions.

An increasing number of chemoselective coupling reactions have beenadvanced for the labeling of full-sized biomolecules. All of thereported methods have their particular strengths and ideal usages, andwith the addition of each technique, new possibilities have arisen forthe generation of complex structures comprising multiple biomolecularcomponents. To add to this list, we have reported a highly efficientoxidative coupling reaction that occurs between anilines and phenylenediamines in the presence of aqueous sodium periodate. This reaction hasshown exceptional chemoselectivity to date, and proceeds rapidly atmicromolar concentrations and at neutral pH. In this report, we applythis method to attach 20-60 copies of DNA aptamers to the surface ofgenome-free viral capsids. The resulting multivalent assemblies bind totyrosine kinase receptors on the surface of Jurkat cells and are readilyendocytosed. Finally, we show that this chemistry can be combined withother bioconjugation methods that could install functional drugmolecules within the carriers. The ability of the oxidative couplingstrategy to prepare these heterobiomolecular structures bodes well forits use in the preparation of many different types of delivery vehicles.

Bacteriophage MS2 provides a readily available scaffold for theconstruction of targeted delivery agents. The protein coat of this virusconsists of 180 sequence-identical monomers that are arranged in ahollow spherical structure. The coat protein monomer can be expressedand self-assembled readily in E. coli, yielding robust, non-toxic andbiodegradable structures that are genome-free. See Carrico, Z. M.;Romanini, D. W.; Mehl, R. A.; Francis, M. B. Chem. Commun. 2008,1205-1207 and hereby incorporated by reference. As MS2 capsids possessthirty-two pores that allow access to the inside of the capsid,selective modification can be achieved on both the interior and theexterior surfaces using orthogonal bioconjugation reactions. (Hooker, J.M.; Kovacs, E. W.; Francis, M. B. Journal of the American ChemicalSociety. 2004, 126, 3718-3719; Kovacs, E. W.; Hooker, J. M.; Romanini,D. W.; Holder, P. G.; Berry, K. E.; Francis, M. B. BioconjugateChemistry. 2007, 18, 1140-1147). In previous reports, we have shown thattyrosine-based chemistry can be used to install F-18 PET tracers(Hooker, J. M.; O'Neil, J. P.; Romanini, D. W.; Taylor, S. E.; Francis,M. Molecular Imaging and Biology. 2008, 10, 182-191) and Gd-based MRIcontrast enhancement agents (Hooker, J. M.; Datta, A.; Botta, M.;Raymond, K. N.; Francis, M. B. Nano Letters. 2007, 7, 2207-2210; Datta,A.; Hooker, J. M.; Botta, M.; Francis, M. B.; Aime, S.; Raymond, K. N.Journal of the American Chemical Society. 2008, 130, 2546-2552) to allowtheir use in imaging applications.

To endow the capsids with specific targeting capabilities, we havedeveloped an efficient synthetic method to attach nucleic acid aptamersto their surfaces. Using the SELEX (Systematic Evolution of Ligands byExponential Enrichment) process, aptamer sequences can be evolved tobind virtually any target, (Tuerk, C.; Gold, L. Science. 1990, 249,505-510; Ellington, A. D.; Szostak, J. W. Nature. 1990, 346, 818-822)and once the composition has been identified, they can be obtainedreadily using automated solid phase synthesis techniques. Theirsynthesis is also amenable to the introduction of modified backbonesthat can improve stability or impart novel functionality. In addition,aptamers can often match or even surpass the specificity and affinity ofantibodies, with the added convenience of smaller size. These qualitiesmake them attractive tools for the development of targeted therapeuticsand imaging platforms with widely varied targeting capabilities.

In order to install oligonucleotide aptamers on the surface of the MS2capsids, we chose a previously reported NaIO₄-mediated oxidativecoupling strategy. This method entails the chemoselective coupling of anN,N-diethyl-N′-acylphenylene diamine moiety to an aniline in thepresence of NaIO₄. In previous studies, sodium periodate has been usedto oxidize the 1,2-diol at the 3′-end of RNA to introduce aldehydefunctionalities, but was not observed to degrade the RNA strand.Although this observation suggests that RNA-based aptamers could be usedwith our method, we chose to proceed with DNA due to its enhancedstability towards hydrolysis during the isolation and handling steps.The aniline coupling partners can be introduced on the exterior surfaceof MS2 capsids either through direct chemical modification or throughthe introduction of an unnatural amino acid, p-aminophenylalanine (paF),into position 19 of the MS2 coat protein using the amber stop codonsuppression system. (Carrico, Z. M.; Romanini, D. W.; Mehl, R. A.;Francis, M. B. Chem. Commun. 2008, 1205-1207). Using the latter route,we have reported previously that the oxidative coupling strategy canintroduce over 100 copies of peptide chains bearing phenylene diaminegroups. For this report, we again used the suppression method to producecapsids bearing the T19paF mutation (MS2-paF19), thus providing 180aniline groups for coupling to the exterior surface. In this case,however, we also introduced an N87C mutation to provide 180 sulfhydrylgroups on the interior surface for cargo installation. The resultingMS2-paF19-N87C double mutant was obtained with a yield of 10 mg/L ofculture.

To develop the reaction conditions, a 20-base DNA sequence, strand A,was first chosen. Starting from an amine-terminated version of thissequence, the N,N-diethyl-N-acylphenylene diamine moiety was easilyintroduced through acylation with an NHS-ester containing precursor.Next, reaction conditions were screened by varying the concentrations ofreacting DNA, sodium periodate, and reaction time (FIG. 25). Optimal DNAcoupling was achieved using 10-20 equivalents (200-400 μM) of the DNAconjugate relative to MS2-paF19 coat protein (20 μM, based on monomer)and 250 equivalents of periodate (5 mM) at room temperature for 1 h. Anadditional increase in the coupling efficiency was observed when thereaction was run at higher concentrations of sodium chloride, which islikely due to the ability of the higher ionic strength to shield thebuildup of negative charge density on the capsid surface as more DNA isattached. For strand A, SDS-PAGE and Coomassie staining, followed byoptical densitometry, indicated that 32% of the capsid monomers had beenmodified with a single strand of DNA, corresponding to 55 strands oneach intact capsid. Longer sequences showed slightly lower conversion,most likely due to increased steric effects as well as electrostaticrepulsion. Following the reaction, the modified capsids could beseparated from excess DNA using either size exclusion chromatography orcentrifugal concentrators with 100 kDa molecular weight cutoffs. As atechnical note, care must be taken to remove all glycerol, ethyleneglycol or other vicinal diols from the samples to prevent them fromreacting with the periodate.

As shown in FIG. 25c,d , the resulting capsids remained intact bytransmission electron microscopy (TEM) and dynamic light scattering(DLS). DLS showed an increase of 10.5±0.7 nm in the hydrodynamicdiameter upon conjugation of strand A to the capsids. When thecomplementary strand was introduced, the diameter increased by anadditional 3.9±1.0 nm, suggesting that the DNA was still capable ofWatson-Crick-Franklin base pairing when conjugated to the exterior ofthe capsid. The ability to base pair also provides good evidence thatthe DNA strands are stable throughout the oxidative coupling conditions.Using denatured capsid monomers, base-pairing of the conjugated DNA wasfurther confirmed by SDS-PAGE using a gel-shift assay (FIG. 25b ).

For functionalization of the interior surface of MS2, standard cysteinebioconjugation was chosen. MS2 contains two native cysteines that haveproven to be inaccessible under normal maleimide bioconjugationconditions. Therefore, a double mutant (MS2-paF19-N87C) was expressed tointroduce a cysteine on the interior surface. The reactivity of thecysteine mutant was shown, where MS2 becomes fluorescently labeled inthe presence of Alexa Fluor 488 maleimide (AF488). Furthermore,MALDI-TOF MS shows near-quantitative conversion to the singly-modifiedproduct. MS2-paF19 capsids lacking the cysteine mutation showed no dyeincorporation under identical conditions.

To finish synthesizing the delivery vehicle shown in FIG. 24, a41-nucleotide DNA aptamer that targets a specific cell surface marker onJurkat cells (strand B) was chosen as the exterior targeting group.Strand B, previously reported as sgc8c, was isolated using a cell-SELEXprocess, and its binding partner was determined to be protein tyrosinekinase 7 (PTK7). PTK7 is a transmembrane protein that is present on thesurface of Jurkat T leukemia cells, as well as many other leukemia celllines, and has been proposed as a potential biomarker for T cell acutelymphoblastic leukemia. Using the oxidative coupling strategy, 20-40copies of diethyl phenylenediamine-labeled strand B were attached toeach capsid, as determined by SDS-PAGE and densitometry analysis (FIG.25a , lane 8). To detect the capsids in cell-binding assays, theinterior was modified with AF488 chromophores as described above priorto DNA attachment.

We tested the targeting specificity of these capsids by incubating themwith Jurkat cells at 37° C. for 30-60 min in culture media. Subsequentanalysis using flow cytometry revealed that samples of cells exposed toMS2 capsids bearing strand B (11 nM in capsids) showed a significantincrease in mean fluorescence intensity compared to background cellularautofluorescence, FIG. 26a . For negative controls, we synthesizedAF488-modified capsids with no exterior modification (AF488-MS2), aswell as ones modified with a 41-nt strand of a randomized sequence (C).Both control capsids did not give rise to an increase in meanfluorescence intensity, confirming the role of the specific aptamersequence in cell-binding.

Having validated the targeting of B-capsids in flow cytometryexperiments, we investigated the cellular internalization of themodified capsids with confocal microscopy. After incubation for 30-60min at 37° C. with Jurkat cells, the presence of capsids labeled withstrand B could be detected as brightly fluorescent dots within thecells, FIG. 3b . Co-staining experiments with fluorescent endocyticmarkers indicated that the B-labeled capsids co-localized withlow-density lipoprotein (LDL) particles, but not transferrin. While bothtransferrin and LDL are known endocytic markers, they traffic throughdifferent pathways once inside the cell. Transferrin has been shown toindicate endosomes that are directed back to the surface through therecycling pathway, while vesicles associated with LDL eventually trafficto lysosomes. In combination with the targeting specificity of theα-PTK7 aptamer, the lysosomal fate of B-capsids is encouraging for thetargeted drug delivery of acid-labile prodrugs that would bepreferentially released upon lysosomal acidification.

For any delivery vehicle composition, the attachment of unprotectedbiomolecular targeting agents will likely be of key importance toachieve tissue specificity. This report demonstrates the utility of achemoselective oxidative coupling reaction for this purpose. Inprinciple, the MS2-based vehicle described herein can now be targeted toany receptor for which a binding aptamer has been determined. For thepurposes of diagnostic imaging it may not be necessary for the capsidsto be internalized; however the observed uptake is envisioned to behighly beneficial for drug delivery applications. In currentexperiments, we are adding anticancer drugs to these carriers, as wellas radionuclides and contrast agents that can be used to determine thelocation of cellular markers in vivo.

General Procedures and Materials.

Unless otherwise noted, all chemicals and solvents were of analyticalgrade and used as received from commercial sources. Analytical thinlayer chromatography (TLC) was performed on EM Reagent 0.25 mm silicagel 60-F254 plates with visualization by ultraviolet (UV) irradiation at254 nm or potassium permanganate stain. All organic solvents wereremoved under reduced pressure using a rotary evaporator.Dichloromethane (CH2Cl2) was distilled under a nitrogen atmosphere fromcalcium hydride. Water (dd-H2O) used in biological procedures or as thereaction solvent was deionized using a NANOpure purification system(Barnstead, USA). 4-(4-diethylamino-phenylcarbamoyl)-butyric acidsuccinimidyl ester was prepared using the previously reported method.1All oligonucleotides were obtained from Integrated DNA Technologies(Coralville, Iowa). Samples were purified by reverse-phase HPLC or NAP-5gel filtration columns (GE Healthcare). Samples were lyophilized using aLAB CONCO Freezone 4.5 (Lab Conco). Lyophilized oligonucleotides werere-suspended in the appropriate buffer and the concentration wasdetermined by measuring the absorbance at 260 nm. All cell culturereagents were obtained from Gibco/Invitrogen Corp (Carlsbad, Calif.)unless otherwise noted. Cell culture was conducted using standardtechniques. Jurkat cells were grown in T-25 culture flasks (Corning,USA) in RPMI Medium 1640 supplemented with 10% (v/v) fetal bovine serum(FBS, HyClone) and 1% penicillin/streptomycin (P/S, Sigma).

Instrumentation and Sample Analysis NMR.

1H and 13C spectra were measured with a Bruker AVQ-400 (400 MHz)spectrometer. Chemical shifts are reported as δ in units of parts permillion (ppm) relative to chloroform-d (δ 7.26, s). Multiplicities arereported as follows: s (singlet), d (doublet), t (triplet), q (quartet),dd (doublet of doublets), p (pentet), m (multiplet), br (broadened), orapp (apparent). Coupling constants are reported as a J value in Hertz(Hz). The number of protons (n) for a given resonance is indicated nH,and is based on spectral integration values.

Mass Spectrometry.

Matrix assisted laser desorption-ionization time-of-flight massspectrometry (MALDI-TOF MS) was performed on a Voyager-DETM system(PerSeptive Biosystems, USA). Prior to MALDI-TOF MS analysis, sampleswere desalted using C18 ZipTip® pipet tips (Millipore, USA).Oligonucleotide samples were co-crystallized using a 3-hydroxypicolinicacid:ammonium citrate solution (45 mg/mL:5 mg/mL in 4.5:5.5 MeCN:ddH2O).For electrospray ionization mass spectrometry (ESI-MS) oligonucleotideconjugates were analyzed using an LTQ Orbitrap XL mass spectrometerequipped with an Ion Max electrospray ionization (ESI) source (ThermoFisher Scientific, Waltham, Mass.). Sample solutions were infused intothe ESI probe at a flow rate of 5 μL/min using a syringe pump. Thevoltages applied to the ion optics were adjusted automatically foroptimum desolvation and transmission of the ions of interest using TunePlus software (version 2.4, Thermo). Mass spectra were recorded in thenegative ion mode over the range m/z=500 to 2000 for a period of twominutes. Mass spectra were processed using Xcalibur software (version4.1, Thermo) and the measured charge state distributions weredeconvoluted using ProMass software (version 2.5 SR-1, Novatia, MonmouthJunction, N.J.). Prior to ESI-MS analysis, oligonucleotides wereprepared as previously described.2 All MS data for oligonucleotides andprotein samples were found to be within 0.1% of the expected values.

High Performance Liquid Chromatography (HPLC).

HPLC was performed on an Agilent 1100 Series HPLC System (AgilentTechnologies, USA). Sample analysis for all HPLC experiments wasachieved with an inline diode array detector (DAD). Both analytical andpreparative reverse-phase HPLC of oligonucleotides was accomplishedusing a C18 stationary phase and a MeCN/100 mM triethylammonium acetate(TEAA, pH=7.0) gradient.

Gel Analyses.

For protein analysis, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) was carried out on a Mini-Protean apparatusfrom Bio-Rad (Hercules, Calif.), following the protocol of Laemmli.3 Allprotein electrophoresis samples were heated for 10 minutes at 100° C. inthe presence of 1,4-dithiothreitol (DTT) to ensure reduction of anydisulfide bonds. Gels were run for 5 minutes at 30V and 70-90 minutes at120V to allow good separation of bands. Commercially available markers(Bio-Rad) were applied to at least one lane of each gel for assignmentof apparent molecular masses. Visualization of protein bands wasaccomplished by staining with Coomassie Brilliant Blue R-250 (Bio-Rad).For fluorescent protein conjugates, visualization was accomplished on aUV backlight. Gel imaging was performed on an EpiChem3 Darkroom system(UVP, USA).

Dynamic Light Scattering.

DLS measurements were obtained using a Malvern Instruments ZetasizerNano ZS, usage courtesy of Jean Fréchet. Data plots and standarddeviations are calculated from an average of three measurements, each ofwhich consists of 10 runs of 45 seconds each. Measurement data arepresented as an intensity plot, which weights larger dimensions by afactor of 106 more than smaller dimensions. Samples taken in 10 mM pH7.0 phosphate buffer.

Transmission Electron Microscopy (TEM).

TEM images were obtained at the UCBerkeley Electron Microscope Lab usinga FEI Tecnai 12 transmission electron microscope with 100 kVaccelerating voltage. TEM grids were prepared by charging carbon-coated,formvar-supported copper mesh grids with argon plasma (40 mA at 0.1 mbarfor 30 s) in a Cressington 108 Auto Sputter Coater. Protein samples wereprepared for TEM analysis by pipetting 5 μL samples onto these grids andallowing them to equilibrate for 3 minutes. The samples were then wickedwith filter paper and rinsed with ddH2O. The grids were then exposed to5 μL of a 1% (w/v) aqueous solution of uranyl acetate for 90 s as anegative stain. After excess stain was removed, the grid was allowed todry in air.

Experimental General Procedure for the Addition of Phenylene Diamine toOligonucleotides.

DNA oligonucleotides were purchased containing a primary amine on the5′-end. A typical reaction is as follows: DNA at a concentration 300 μMis reacted with 4-(4-diethylamino-phenylcarbamoyl)-butyric acidsuccinimidyl ester (60-120 eq) in 1:1 solution of DMF and 50 mM pH 8.0phosphate buffer. The reaction mixture is briefly vortexed and thenallowed to react at rt for 2 h. Either RPHPLC or commercially availablegel filtration columns can be used to purify the small molecule fromDNA, following the commercially provided protocol. Followingpurification, DNA is lyophilized and then re-suspended in the desiredbuffer. Concentration is determined by measuring the absorbance at 260nm. The sequence identities of A, B, and C are as follows:

A: (SEQ ID NO: 7) 5′-TCATACGACTCACTCTAGGGA-3′ B: (SEQ ID NO: 8)5′-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3′ C: (SEQ ID NO: 9)5′-CCCTAGAGTGAGTCGTATGACCCTAGAGTGAGTCGTATGAA-3′

General Procedure for DNA Conjugation to MS2.

An eppendorf tube is charged with either MS2-paF19 or MS2-paF19-N87C (20μM), phenylene diamine-containing oligonucleotide (200-400 μM), andNaIO4 (5 mM). The reaction is carried out in 50 mM pH 7.0 phosphatebuffer containing 150 mM NaCl. The reaction is briefly vortexed andallowed to react at rt for 1 h. After an hour, for a 50 μL reaction, thereaction is quenched by the addition of 5 μL of 500 mMtris(2-carboxyethyl)phosphine hydrochloride (TCEP). For purification,the sample is first bufferexchanged by gel filtration (NAP-5) into thedesired buffer. The excess DNA is then removed by successive centrifugalfiltration using 100 k molecular weight cutoff filters (Millipore).

Cloning and Expression of MS2 Mutants.

The pBAD-MS2-paF19 plasmid production and growth has been previouslyreported.1 We would like to thank the Peter Schultz lab (ScrippsResearch Institute, LaJolla, Calif.) for the tRNA- andtRNA-synthetase-encoding plasmids necessary for p-aminophenylalanine(paF) incorporation. Position 87 was mutated into a cysteine using thefollowing forward and reverse primers: Forward:5′-AGCCGCATGGCGTTCGTACTTATGTATGGAACTAACCATTC-3′ (SEQ ID NO:25); Reverse:5′-GAATGGTTAGTTCCATACATAAGTACGAACGCCATGCGGCT-3′ (SEQ ID NO:26). Growthand purification of MS2-paF19-N87C was identical to that of MS2-paF19,although a lower yield was obtained for MS2-paF19-N87C (˜1-10 mg/L ascompared to ˜20 mg/L for MS2-paF19).

Dual-Surface Modification of MS2-paF19-N87C.

MS2-paF19-N87C is first modified on the interior cysteine. For thecysteine alkylation reaction, Alexa Fluor 488 maleimide (Invitrogen) (15μL of a 19 mM solution in DMSO) is added to MS2-paF19-N87C (285 μL of a100 μM solution in 10 mM pH 7.2 phosphate buffer). The reaction isbriefly vortexed and allowed to react at rt for 1 h. Excess smallmolecule is removed by gel filtration (NAP-5) and the remaining proteinwas concentrated using centrifugal filtration. It is important to notethat centrifugal filters were prerinsed before use as we found this toprevent problems with the oxidative coupling step. The exteriormodification was performed as described above.

Flow Cytometry of Fluorescent Capsids.

Flow cytometry analysis was acquired on a FACSCalibur flow cytometer (BDBiosciences, USA) using a standard 488 Ar laser. Data were collected forat least 10,000 live cells for all experiments. Jurkat cells (1×106cells in 250 μL) were treated with fluorescent capsids (2 μM) in culturemedia and incubated either on ice or at 37° C. for 30-60 min. Afterincubation, the cells were washed twice with 1 mL of fresh media, andthen analyzed on the flow cytometer.

Confocal Microscopy.

Confocal fluorescence imaging was carried out on a Zeiss LSM510 META/NLOAxioimager using a 63× Achroplan IR oil-immersion objective lens. Doublelabeling of Jurkat cells was performed by incubating cells with modifiedMS2 (20 μM) and DiILDL (15 μg/mL, Invitrogen) or Alexa Fluor594-Transferrin (25 μg/mL, Invitrogen) for 30-60 min at 37° C. ModifiedMS2 (fluorescently-labeled with Alexa Fluor 488) was excited at 488 nmand emission was collected between 495-530 nm. Both DiI-LDL andAF594-Transferrin were excited with a 543 nm line and emission wascollected between 590-625 nm and 590-655 nm, respectively.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

SEQUENCES SEQ ID NO: 1 GTA ACG ATC CAG CTG TCA CT SEQ ID NO: 2AGT GAC AGC TGG ATC GTT AC SEQ ID NO: 3 TCA TAC GAC TCA CTC TAG GGSEQ ID NO: 4 CCC TAG AGT GAG TCG TAT GA SEQ ID NO: 5ACT GAC TGA CTG ACT GAC TG SEQ ID NO: 6 CAG TCA GTC AGT CAG TCA GTSEQ ID NO: 7 TCATACGACTCACTCTAGGGA SEQ ID NO: 8ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA SEQ ID NO: 9CCCTAGAGTGAGTCGTATGACCCTAGAGTGAGTCGTATGAA

1.-12. (canceled)
 13. A method of preparing a conjugate of a live celland a nucleic acid moiety, wherein (i) the cell has a surface comprisinga native functional group that is not an azide-modified sugar, and (ii)the cell has no cell wall, the method comprising: contacting the cellwith an activated nucleic acid moiety under conditions such that theactivated nucleic acid moiety reacts with the native functional group tocovalently link the nucleic acid directly to the native functionalgroup. 14.-30. (canceled)
 31. The method of claim 13, wherein the cellis a primary cell.
 32. The method of claim 13, wherein the cell is amammalian cell.
 33. The method of claim 13, wherein the cell is a stemcell.
 34. The method of claim 13, wherein the native functional groupcomprises an amino acid selected from the group consisting of lysine,cysteine, tyrosine, threonine, serine, aspartic acid, glutamic acid andtryptophan.
 35. The method of claim 13, wherein the native functionalgroup comprises lysine.
 36. The method of claim 13, wherein the nucleicacid moiety comprises a member selected from the group consisting of anoligonucleotide, DNA, RNA, PNA and an aptamer.
 37. The method of claim13, wherein the nucleic acid moiety comprises single-stranded DNA. 38.The method of claim 13, wherein the nucleic acid moiety comprises fromabout 10 to about 200 nucleic acids.
 39. The method of claim 13, whereinthe nucleic acid moiety comprises an aptamer.
 40. The method of claim13, wherein the nucleic acid moiety comprises a linker.
 41. The methodof claim 13, wherein the conjugate comprises a mammalian cell comprisinglysine on the cell surface; and a single-stranded deoxy-nucleic acidcovalently linked to the lysine via an amide.
 42. A method of preparinga conjugate of a live cell and a nucleic acid moiety, wherein (i) thecell has a surface comprising a native functional group comprising anamine, and (ii) the cell has no cell wall, the method comprisingcontacting the cell with an activated nucleic acid moiety underconditions such that the activated nucleic acid moiety reacts with aprimary amine of the native functional group to form a covalent bond.43. The method of claim 42, wherein the cell is a primary cell.
 44. Themethod of claim 42, wherein the cell is a mammalian cell.
 45. The methodof claim 42, wherein the cell is a stem cell.
 46. The method of claim42, wherein the cell has not undergone metabolic engineering tointroduce an azide modified sugar.
 47. The method of claim 42, whereinthe native functional group comprises an amino acid.
 48. The method ofclaim 47, wherein the amino acid is selected from the group consistingof lysine, cysteine, tyrosine, threonine, serine, aspartic acid,glutamic acid and tryptophan.
 49. The method of claim 42, wherein thenative functional group comprises lysine.
 50. The method of claim 42,wherein the nucleic acid moiety comprises a member selected from thegroup consisting of an oligonucleotide, DNA, RNA, PNA and an aptamer.51. The method of claim 42, wherein the nucleic acid moiety comprisessingle-stranded DNA.
 52. The method of claim 42, wherein the nucleicacid moiety comprises from about 10 to about 200 nucleic acids.
 53. Themethod of claim 42, wherein the nucleic acid moiety comprises anaptamer.
 54. The method of claim 42, wherein the nucleic acid moietycomprises a linker.
 55. The method of claim 42, wherein the conjugatecomprises a mammalian cell comprising lysine on the cell surface; and asingle-stranded deoxy-nucleic acid covalently linked to the lysine viaan amide.
 56. A method of preparing a conjugate of a live cell and anucleic acid moiety, wherein the cell has a surface comprising a nativefunctional group comprising an amino acid selected from the groupconsisting of lysine, cysteine, tyrosine, threonine, serine, asparticacid, glutamic acid, and tryptophan, and wherein the cell has no cellwall, the method comprising: contacting the cell with an activatednucleic acid moiety under conditions such that the nucleic acid moietyreacts with the amino acid to form a covalent bond.
 57. The method ofclaim 56, wherein the cell is a primary cell.
 58. The method of claim56, wherein the cell is a mammalian cell.
 59. The method of claim 56,wherein the cell is a stem cell.
 60. The method of claim 56, wherein thecell has not undergone metabolic engineering to introduce an azidemodified sugar.
 61. The method of claim 56, wherein the nativefunctional group comprises lysine.
 62. The method of claim 56, whereinthe nucleic acid moiety comprises a member selected from the groupconsisting of an oligonucleotide, DNA, RNA, PNA and an aptamer.
 63. Themethod of claim 56, wherein the nucleic acid moiety comprisessingle-stranded DNA.
 64. The method of claim 56, wherein the nucleicacid moiety comprises from about 10 to about 200 nucleic acids.
 65. Themethod of claim 56, wherein the nucleic acid moiety comprises anaptamer.
 66. The method of claim 56, wherein the nucleic acid moietycomprises a linker.